Low acidic species compositions and methods for producing and using the same using displacement chromatography

ABSTRACT

The present invention relates to low acidic species (AR) compositions comprising a protein, e.g., an antibody, or antigen-binding portion thereof, and methods for producing such low AR compositions using displacement chromatography. Methods for using such compositions to treat a disorder, e.g., a disorder in which TNFα is detrimental, are also provided.

RELATED APPLICATION INFORMATION

This application is a continuation of U.S. application Ser. No.14/077,576, filed on Nov. 12, 2013, which in turn claims priority toU.S. Provisional Application Ser. No. 61/892,833, filed on Oct. 18, 2013and U.S. application Ser. No. 13/803,808, filed on Mar. 14, 2013, theentire contents of each of which are expressly incorporated herein byreference.

BACKGROUND OF THE INVENTION

The production of compositions comprising proteins for biopharmaceuticalapplications involves the use of upstream process technologies (e.g.,cell culture) and downstream process technologies (e.g., proteinpurification) that are known to produce proteins exhibiting varyinglevels of protein variants and impurities within the composition. Suchprotein variants include, but are not limited to, the presence of chargevariants (e.g., basic variants and acidic species, including variants)and process-related impurities. For example, in monoclonal antibody(mAb) preparations, charge variants can be detected by various methods,such as ion exchange chromatography, for example, WCX-10 HPLC (a weakcation exchange chromatography) or IEF (isoelectric focusing). Becauseof their similar chemical characteristics to the antibody productmolecules of interest, reduction of charge variants is a challenge inmonoclonal antibody production.

Reduction of charge variants and/or product- or process-relatedimpurities is particularly advantageous in the context of commerciallyproduced recombinant biotherapeutics, as they have the potential toimpact numerous product characteristics, including, but not limited to,product stability, product safety and product efficacy. Accordingly,there remains a need in the art for low acidic species compositions andhigh-efficiency methods of producing protein compositions, e.g.,antibodies, having low levels of acidic species.

SUMMARY OF THE INVENTION

The present invention is based on the optimization of displacementchromatography process technologies for protein production, e.g.,production of antibodies or antigen-binding portions thereof, resultingin the production of compositions comprising proteins that comprise lowpercentages of acidic species. These low acidic species compositionshave improved therapeutic efficacy and improved biological properties,for example, increased cartilage tissue penetration, reduced cartilagedestruction, reduced synovial proliferation, reduced bone erosion,increased protection against the development of arthritis as measured byarthritic scores and/or histopathology scores, reduced cellinfiltration, reduced proteoglycan loss, reduced chondrocyte death,and/or increased TNFα affinity, as compared to a non-low acidic speciescomposition.

Displacement chromatography is a chromatographic separation technologythat involves the use of a displacer molecule to aid in the separationof a mixture, e.g., an antibody-containing solution derived from cellculture harvest. The displacer molecule is conventionally selected tohave a higher affinity for the stationary phase (i.e., thechromatographic support) as compared to the components present in thematerial to be separated. Due to its higher affinity, the displacermolecule competes with protein mixture components for the binding siteson the stationary phase. Under appropriate conditions, the displacerinduces the components of the mixture to develop into consecutive zonesof concentrated and purified species in the order of decreasing bindingaffinity ahead of the displacer front. This ordered displacement of thecomponents of the mixture results in the formation of a so-called“displacement train.” In contrast to traditional elution modechromatography, the displacement process takes advantage of thenonlinearity of the adsorption isotherm, allowing for higher columnloading levels without compromising the purity and recovery of thecomponent of interest. Finally, washing of the displacement train withthe displacing buffer from the column allows for the component ofinterest to be isolated by collecting (and pooling if necessary) theproper fraction(s) of the displaced eluate.

Accordingly, in one aspect, the invention provides a method forproducing a low acidic species composition comprising an antibody, orantigen-binding portion thereof, by contacting a first sample comprisingthe antibody, or antigen-binding portion thereof, with a chromatographymedia, wherein the antibody, or antigen-binding portion thereof, bindsto the chromatography media; displacing the antibody, or antigen-bindingportion thereof, bound to the chromatography media with a displacingbuffer comprising at least one displacer molecule; and collecting achromatography sample, wherein the chromatography sample comprises acomposition of the antibody, or antigen-binding portion thereof, whichcontains less than about 10% acidic species, thereby producing a lowacidic species composition comprising an antibody, or antigen-bindingportion thereof.

In one embodiment, the chromatography media is selected from the groupconsisting of anion exchange adsorbent material, cation exchangeadsorbent material and mixed mode media. In another embodiment, thecation exchange (CEX) adsorbent material is selected from the groupconsisting of a CEX resin and a CEX membrane adsorber. In anotherembodiment, the CEX resin is Poros XS resin.

In another embodiment, the chromatography media is a mixed mode mediacomprising cation exchange (CEX) and hydrophobic interaction functionalgroups. In one embodiment, the mixed mode media is Capto MMC resin. Inanother embodiment, the mixed mode media is selected from the groupconsisting of a CEX-based mixed mode resin and a CEX-based mixed modemembrane adsorber. In another embodiment, the mixed mode media isselected from the group consisting of CaptoMMC ImpRes, Nuvia cPrime, andToyopearl Trp-650M resins.

In one embodiment, the pH of the displacing buffer is lower than theisoelectric point of the antibody, or antigen-binding portion thereof.In another embodiment, the pH of the displacing buffer is in the rangeof about 6.0 to about 8.0.

In one embodiment, the displacing buffer carries positive charge andwherein the concentration of the displacer in the displacing buffer isat least about 0.1 mM. In another embodiment, the displacer is aquaternary ammonium salt and the concentration of the displacer in thedisplacing buffer about 0.1 mM to about 10 mM. In another embodiment,the displacer is protamine sulfate and the concentration of theprotamine sulfate in the displacing buffer is about 0.1 mM to 5 aboutmM.

In one embodiment, the conductivity of the displacing buffer is about 2mS/cm to about 20 mS/cm. In another embodiment, the chromatography mediais in a column, wherein the column length is in the range of about 10 cmto about 30 cm, and wherein flow residence time is in the range of about5 min to about 25 min.

In one embodiment, one displacing buffer is used.

In another embodiment, a first displacing buffer and a second displacingbuffer are used, and wherein the first and second displacing bufferscomprise different concentrations of displacer. In one embodiment, thefirst displacing buffer comprises a lower displacer concentration ofdisplacer than the second displacing buffer. In one embodiment, thefirst displacing buffer comprises about 0.5 mM Expell SP1™. In anotherembodiment, the first displacing buffer comprises about 0.25 mMprotamine sulfate.

In one embodiment, the method is run in a two-step displacementchromatography mode.

In another embodiment, the method is run in a multiple-step displacementchromatography mode or a linear displacement chromatography mode.

In one embodiment, the chromatography sample comprises a reduced levelof host cell proteins as compared to the first sample. In anotherembodiment, the chromatography sample comprises a reduced level of oneor more of charge variants, structure variants or fragmentation variantsas compared to the first sample. In one embodiment, the chromatographysample comprises a reduced level of acidic species region AR1, andwherein the charge variants comprise deamidation variants, glycationvariants, afucosylation variants, MGO variants or citric acid variants.In another embodiment, the chromatography sample comprises a reducedlevel of the acidic species region AR1, and wherein the structurevariants comprise glycosylation variants or acetonation variants. In oneembodiment, the chromatography sample comprises a reduced level of theacidic species region AR1, and wherein the fragmentation variantscomprise Fab fragment variants, C-terminal truncation variants orvariants missing a heavy chain variable domain. In another embodiment,the chromatography sample comprises a reduced level of the acidicspecies region AR2, and wherein the charge variants comprise deamidationvariants or glycation variants.

In another embodiment, the chromatography sample comprises a reducedlevel of basic species as compared to the first sample. In oneembodiment, the reduced level of basic species comprise a reduced levelof a lysine species Lys 0 as compared to the first sample.

In another embodiment, the chromatography sample comprises a reducedlevel of aggregates as compared to the first sample. In anotherembodiment, the chromatography sample comprises a reduced level ofantibody fragments as compared to the first sample.

In one embodiment, the antibody, or antigen-binding portion thereof, isan anti-TNFα antibody, or antigen-binding portion thereof. In oneembodiment, the antibody, or antigen-binding portion thereof, hasdissociates from human TNFα with a K_(d) of about 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ S⁻¹ or less. In another embodiment, theanti-TNFα antibody, or antigen-binding portion thereof, comprises alight chain variable region (LCVR) having a CDR1 domain comprising theamino acid sequence of SEQ ID NO: 7, a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 5, and a CDR3 domain comprising the aminoacid sequence of SEQ ID NO: 3; and a heavy chain variable region (HCVR)having a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8,a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and aCDR3 domain comprising the amino acid sequence of SEQ ID NO: 4. Inanother embodiment, the anti-TNFα antibody, or antigen-binding portionthereof, comprises a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 1 and a heavy chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 2. Inanother embodiment, the anti-TNFα antibody, or antigen-binding portionthereof, is adalimumab, or an antigen binding-portion thereof.

In another aspect, the present invention provides a low acidic species(low AR) composition comprising an antibody, or antigen-binding portionthereof, where the composition comprises about 15% or less AR. In oneaspect of this embodiment, the low AR composition comprises about 14% orless AR, 13% or less AR, 12% or less AR, 11% or less AR, 10% or less AR,9% or less AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or lessAR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR, 2.5%or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% orless AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or lessAR, 1.2% or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR,0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR, 0.4%or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or 0.0%AR, and ranges within one or more of the preceding. In one aspect ofthis embodiment, the present invention provides a low AR compositioncomprising an antibody, or antigen-binding portion thereof, where thecomposition comprises about 0.0% to about 10% AR, about 0.0% to about 5%AR, about 0.0% to about 4% AR, about 0.0% to about 3% AR, about 0.0% toabout 2% AR, about 3% to about 5% AR, about 5% to about 8% AR, or about8% to about 10% AR, or about 10% to about 15% AR, and ranges within oneor more of the preceding.

In one embodiment, the low AR composition comprises a first acidicspecies region (AR1) and a second acidic species region (AR2). In oneaspect of this embodiment, the low AR composition comprises about 0.1%or less AR1 and about 3% or less AR2, or about 0.0% AR1 and about 1.4%or less AR2. In a related embodiment, the low AR composition comprisesabout 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% or lessAR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1,7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% orless AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or lessAR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or lessAR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or lessAR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or lessAR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or lessAR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1,and ranges within one or more of the preceding. In one aspect of thisembodiment, the present invention provides a low AR compositioncomprising an antibody, or antigen-binding portion thereof, where thecomposition comprises about 0.0% to about 10% AR1, about 0.0% to about5% AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% AR1, about0.0% to about 2% AR1, about 3% to about 5% AR1, about 5% to about 8%AR1, or about 8% to about 10% AR1, or about 10% to about 15% AR1, andranges within one or more of the preceding.

In one aspect of this embodiment, the low AR composition comprises about15% or less 3.5% or less AR2, 14% or less AR2, 13% or less AR2, 12% orless AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% or lessAR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or less AR2,4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2, 2%or less AR2, about 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%,1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2, 1.6% or less AR2,1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2,1.1% or less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2,0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2,0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, andranges within one or more of the preceding. In one aspect of thisembodiment, the present invention provides a low AR compositioncomprising an antibody, or antigen-binding portion thereof, where thecomposition comprises about 0.0% to about 10% AR2, about 0.0% to about5% AR2, about 0.0% to about 4% AR2, about 0.0% to about 3% AR2, about0.0% to about 2% AR2, about 3% to about 5% AR2, about 5% to about 8%AR2, or about 8% to about 10% AR2, or about 10% to about 15% AR2, andranges within one or more of the preceding 0%, 0.9%, 0.8%, 0.7%, 0.6%,0.5%, 0.4%, 0.3%, 0.2% or 0.1% AR2. In another embodiment, the low ARcomposition comprises about 0% AR2.

In another embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab, comprises about 1.4% or less AR. For example,in one aspect of this embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab comprising about 1.4% or less AR can compriseabout 0.0% AR1 and about 1.4% or less AR2.

In one embodiment, the acidic species in the low AR composition compriseone or more variants selected from the group consisting of chargevariants, structure variants, aggretation variants and fragmentationvariants. For example, in one embodiment, the charge variants in the lowAR composition are AR1 species and comprise, for example, deamidationvariants, glycation variants, afucosylation variants, MGO variants orcitric acid variants. In another embodiment, the structure variants inthe low AR composition are AR1 species and comprise, for example,glycosylation variants or acetonation variants. In still anotherembodiment, the fragmentation variants in the low AR composition are AR1and comprise, for example, Fab fragment variants, C-terminal truncationvariants or variants missing a heavy chain variable domain.

In another embodiment, the acidic species in the low AR composition areAR2 variants, and comprise, for example, charge variants such asdeamidation variants or glycation variants.

In another aspect, the present invention provides compositionscomprising an antibody, or antigen-binding portion thereof, wherein thecomposition is substantially free of acidic species such asprocess-related impurities, including, for example, host cell proteins(HCPs), host cell nucleic acids, chromatographic materials, and/or mediacomponents, as well as product related impurities such as aggregatesetc.

In one embodiment, the antibody, or antigen-binding portion thereof, ofthe compositions disclosed herein is an anti-TNFα antibody, orantigen-binding portion thereof. For example, in one aspect of thisembodiment, the anti-TNFα antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(d) of about 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ S⁻¹ or less. In another aspect of thisembodiment, the anti-TNFα antibody, or antigen-binding portion thereof,comprises a light chain variable region (LCVR) having a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 7, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3; and a heavy chainvariable region (HCVR) having a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4.

In still another aspect of this embodiment, the anti-TNFα antibody, orantigen-binding portion thereof, comprises a light chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 2. In yet another aspect of this embodiment, the anti-TNFαantibody, or antigen-binding portion thereof, is adalimumab, or anantigen binding-portion thereof.

In one embodiment, the low AR composition of the invention comprisesadalimumab, and has a percentage of AR that is not the same as thepercentage of AR present in adalimumab formulated as HUMIRA® ascurrently approved and described in the “Highlights of HUMIRA®Prescribing Information” for HUMIRA® (adalimumab) Injection (RevisedJanuary 2008), the contents of which are hereby incorporated herein byreference.

In another embodiment, the low AR composition of the invention comprisesadalimumab, and has a percentage of AR that is lower than the percentageof AR present in adalimumab formulated as HUMIRA® as currently approvedand described in the “Highlights of HUMIRA® Prescribing Information” forHUMIRA® (adalimumab) Injection (Revised January 2008), the contents ofwhich are hereby incorporated herein by reference.

In another embodiment, the present invention provides low ARcompositions comprising an anti-TNFα antibody, or antigen-bindingportion thereof, comprising a light chain variable region (LCVR) havinga CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3domain comprising the amino acid sequence of SEQ ID NO: 3; and a heavychain variable region (HCVR) having a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, wherein the composition comprises less thanabout 10% AR. In one aspect of this embodiment, the anti-TNFα antibody,or antigen-binding portion thereof, comprises a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 1 anda heavy chain variable region comprising the amino acid sequence setforth in SEQ ID NO: 2, wherein the composition comprises less than about10% AR. In another aspect of this embodiment, the anti-TNFα antibody, orantigen-binding portion thereof, is adalimumab, or an antigenbinding-portion thereof, and the composition comprises less than about10% AR. In one aspect of this embodiment, the low AR compositioncomprising an anti-TNFα antibody, or antigen-binding portion thereof,comprises about 0.1% or less AR1 and about 3% or less AR2, or about 0.0%AR1 and about 1.4% or less AR2.

In one embodiment, the acidic species in the low AR compositioncomprising an antibody, or antigen-binding portion thereof (e.g., ananti-TNFα antibody, or antigen binding portion thereof, such asadalimumab) comprise one or more variants selected from the groupconsisting of charge variants, structure variants and fragmentationvariants. For example, in one aspect of this embodiment, the chargevariants in the low AR composition are AR1 species and comprise, forexample, deamidation variants, glycation variants, afucosylationvariants, methylglyoxal (MGO) variants or citric acid variants. Forexample, when the low AR composition comprises adalimumab, thedeamidation variants can result from deamidation occurring at asparagineresidues comprising Asn393 and Asn329 of adalimumab and at glutamineresidues comprising Gln3 and Gln6. In another aspect of this embodiment,when the low AR composition comprises adalimumab, the glycation variantscan result from glycation occurring at Lys98 and Lys151 of adalimumab.

In another aspect of this embodiment, the structure variants in the lowAR composition comprising an antibody, or antigen-binding portionthereof (e.g., an anti-TNFα antibody, or antigen binding portionthereof, such as adalimumab) are AR1 species and comprise, for example,glycosylation variants or acetonation variants.

In still another aspect of this embodiment, the fragmentation variantsin the low AR composition comprising an antibody, or antigen-bindingportion thereof (e.g., an anti-TNFα antibody, or antigen binding portionthereof, such as adalimumab), are AR1 species and comprise, for example,Fab fragment variants, C-terminal truncation variants or variantsmissing a heavy chain variable domain.

In another embodiment, the acidic species in the low AR compositioncomprising an antibody, or antigen-binding portion thereof (e.g., ananti-TNFα antibody, or antigen binding portion thereof, such asadalimumab), are AR2 species, and comprise charge variants, such asdeamidation variants or glycation variants. For example, when the low ARcomposition comprises adalimumab, the deamidation variants can resultfrom deamidation occurring at asparagine residues comprising Asn393 andAsn329 of adalimumab and at glutamine residues comprising Gln3 and Gln6.In another aspect of this embodiment, when the low AR compositioncomprises adalimumab, the glycation variants result from glycationoccurring at Lys98 and Lys151 of adalimumab.

In one embodiment, the percent of acidic species in a low AR compositionis determined using ion exchange chromatography, for example WCX-10HPLC. In another aspect of this embodiment, the percent acidic speciesin a low AR composition is determined using isoelectric focusing (IEF).

In one embodiment, the low AR compositions of the invention compriseproduct preparation-derived acidic species. For example, in one aspectof this embodiment, the acidic species are cell culture-derived acidicspecies. In another aspect of this embodiment, the acidic species of thelow AR compositions are storage-derived acidic species which areprimarily generated when stored under process, intermediate or shelfstorage conditions prior to use.

In still another embodiment, the invention provides low AR compositionsthat further comprise a pharmaceutically acceptable carrier.

In another aspect, the present invention provides methods for treating asubject having a disorder in which TNFα is detrimental, by administeringto the subject a low AR composition of the invention, e.g., a low ARadalimumab composition, thereby treating the subject having a disorderin which TNFα is detrimental. In one aspect of this embodiment, thedisorder in which TNFα is detrimental is selected from the groupconsisting of rheumatoid arthritis (RA), psoriasis, psoriatic arthritis,ankylosing spondylitis, juvenile idiopathic arthritis (JIA), ulcerativecolitis, Crohn's Disease, active axial spondyloarthritis (active axSpA)and non-radiographic axial spondyloarthritis (nr-axSpA).

The present invention is further illustrated by the following detaileddescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts (a) comparison of a desired and an undesired displacementchromatogram for Adalimumab on Poros XS resin using Expell SP1™; (b)charge variants distribution in eluate fractions derived from theundesired displacement chromatography process for Adalimumab (Poros XSresin & Expell SP1™)

FIG. 2 depicts CEX-HPLC chromatograms of Expell SP1™-displacedAdalimumab sample fractions.

FIG. 3 depicts the separation of Adalimumab charge variants by Poros XSdisplacement chromatography using Expell SP1™.

FIG. 4 depicts the reduction of acidic species level in Adalimumab byPoros XS displacement chromatography using Expell SP1™.

FIG. 5 depicts the effect of Expell SP1™ concentration on acidic speciesreduction in Adalimumab by Poros XS displacement chromatography.

FIG. 6 depicts the effect of pH on acidic species reduction inAdalimumab by Poros XS displacement chromatography using Expell SP1™.

FIG. 7 depicts the reduction of acidic species level in Adalimumab byPoros XS two-step displacement chromatography using Expell SP1™.

FIG. 8 depicts the separation of Adalimumab size variants by Poros XSdisplacement chromatography using Expell SP1™.

FIG. 9 depicts the separation of HCP in Adalimumab by Poros XSdisplacement chromatography using Expell SP1™.

FIG. 10 depicts the separation of Adalimumab charge variants by Poros XSdisplacement chromatography using protamine sulfate.

FIG. 11 depicts the reduction of acidic species in Adalimumab by PorosXS displacement chromatography using protamine sulfate.

FIG. 12 depicts the effect of protamine sulfate concentration on acidicspecies reduction in Adalimumab by Poros XS displacement chromatography.

FIG. 13 depicts the effect of pH on acidic species reduction inAdalimumab by Poros XS displacement chromatography using protaminesulfate.

FIG. 14 depicts the reduction of acidic species in Adalimumab by PorosXS two-step displacement chromatography using protamine sulfate.

FIG. 15 depicts the reduction of acidic species in Adalimumab on PorosXS using protamine sulfate linear gradient displacement chromatography.

FIG. 16 depicts the separation of Adalimumab size variants by Poros XSdisplacement chromatography using protamine sulfate.

FIG. 17 depicts the separation of mAb X charge variants by Poros XSdisplacement chromatography using Expell SP1™.

FIG. 18 depicts the reduction of acidic species in mAb X by Poros XSdisplacement chromatography using Expell SP1™.

FIG. 19 depicts the effect of Expell SP1™ concentration on acidicspecies reduction in mAb X by Poros XS displacement chromatography.

FIG. 20 depicts the reduction of acidic species in mAb X by Poros XStwo-step displacement chromatography using Expell SP1™

FIG. 21 depicts the separation of mAb X charge variants by Poros XSdisplacement chromatography using protamine sulfate.

FIG. 22 depicts the reduction of acidic species in mAb X by Poros XSdisplacement chromatography using protamine sulfate.

FIG. 23 depicts the effect of protamine sulfate concentration on acidicspecies reduction in mAb X by Poros XS displacement chromatography.

FIG. 24 depicts the reduction of acidic species in mAb X by Poros XStwo-step displacement chromatography using protamine sulfate.

FIG. 25 depicts the separation of mAb X size variants by Poros XSdisplacement chromatography using protamine sulfate.

FIG. 26 depicts the separation of mAb Y charge variants by Poros XSdisplacement chromatography using Expell SP1™.

FIG. 27 depicts the reduction of acidic species in mAb Y by Poros XSdisplacement chromatography using Expell SP1™.

FIG. 28 depicts the separation of Adalimumab charge variants by CaptoMMC displacement chromatography using protamine sulfate.

FIG. 29 depicts the reduction of acidic species in Adalimumab by CaptoMMC displacement chromatography using protamine sulfate.

FIG. 30 depicts the separation of mAb X charge variants by Capto MMCdisplacement chromatography using protamine sulfate.

FIG. 31 depicts the reduction of acidic species in mAb X by Capto MMCdisplacement chromatography using protamine sulfate.

FIG. 32 depicts the effect of pH on acidic species reduction in mAb X byCapto MMC displacement chromatography.

FIG. 33 depicts the AR Growth at 25° C. of low and high AR containingsamples.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification and optimization ofdisplacement chromatography technologies for protein production, e.g.,production of proteins, e.g., antibodies or antigen-binding portionsthereof, e.g., adalimumab, resulting in compositions that comprise lowpercentages of protein charge variants, e.g., acidic species (AR), andbasic species, and/or low levels of product- and process-relatedimpurities (e.g., aggregates, fragments, host cell proteins and mediacomponents). In one embodiment, the displacement chromatography methodsof the invention can surprisingly be used in large-scale proteinpurification processes due to superior resolution of closely relatedspecies using practically relevant chromatography resins and conditions.In one embodiment, the displacement chromatography methods of theinvention can surprisingly be used in large-scale protein purificationprocesses due to reduced buffer volume for a given separation, therebyproviding improved process efficiency.

The compositions of the present invention exhibit increased therapeuticefficacy when administered to a subject. For example, compositionscomprising anti-TNFα antibodies, or antigen binding portions thereof,comprising low AR are capable of increased therapeutic efficacy in thetreatment and prevention of a disorder in which TNFα is detrimental,e.g., rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA),psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,and ulcerative colitis. Accordingly, the instant invention providescompositions comprising proteins that comprise low AR and/or low levelsof product- and process-related impurities, and methods for producingand using the same.

In one embodiment, the low AR compositions of the invention compriseabout 15% or less AR, 14% or less AR, 13% or less AR, 12% or less AR,11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or lessAR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5%or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9% or lessAR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR,1.4% or less AR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1%or less AR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% orless AR, 0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or lessAR, 0.1% or less AR, or 0.0% AR, and ranges within one or more of thepreceding. In one aspect of this embodiment, the low AR compositions ofthe invention comprise about 0.0% to about 10% AR, about 0.0% to about5% AR, about 0.0% to about 4% AR, about 0.0% to about 3% AR, about 0.0%to about 2% AR, about 3% to about 5% AR, about 5% to about 8% AR, orabout 8% to about 10% AR, or about 10% to about 15% AR, and rangeswithin one or more of the preceding.

In another embodiment, the low AR composition comprises a first acidicspecies region (AR1) and a second acidic species region (AR2). In oneaspect of this embodiment, the low AR composition comprises about 0.1%or less AR1 and about 3% or less AR2. In another aspect of thisembodiment, the low AR composition comprises about 0.0% AR1 and about1.4% or less AR2.

In another aspect of this embodiment, the low AR composition comprisesabout 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% or lessAR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1,7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% orless AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or lessAR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or lessAR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or lessAR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or lessAR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or lessAR1 or less, 0.3% or less AR1 or less, 0.2% or less AR1 or less, 0.1% orless AR1, or 0.0% AR1, and ranges within one or more of the preceding.In one aspect of this embodiment, the low AR compositions of theinvention comprise about 0.0% to about 10% AR1, about 0.0% to about 5%AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% AR1, about 0.0%to about 2% AR1, about 3% to about 5% AR1, about 5% to about 8% AR1, orabout 8% to about 10% AR1, or about 10% to about 15% AR1, and rangeswithin one or more of the preceding.

In yet another aspect of this embodiment, the low AR compositioncomprises about 15% or less AR2, 14% or less AR2, 13% or less AR2, 12%or less AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% orless AR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or lessAR2, 4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2,2% or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2,1.6% or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2,1.2% or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2,0.8% or less AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2,0.4% or less AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2,or 0.0% AR2, and ranges within one or more of the preceding. In oneaspect of this embodiment, the low AR compositions of the inventioncomprise about 0.0% to about 10% AR2, about 0.0% to about 5% AR2, about0.0% to about 4% AR2, about 0.0% to about 3% AR2, about 0.0% to about 2%AR2, about 3% to about 5% AR2, about 5% to about 8% AR2, or about 8% toabout 10% AR2, or about 10% to about 15% AR2, and ranges within one ormore of the preceding.

In another embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab, comprises about 1.4% or less AR. For example,in one aspect of this embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab comprising about 1.4% or less AR comprisesabout 0.0% AR1 and about 1.4% or less AR2.

In one embodiment, the protein is an antibody or antigen binding portionthereof, such as the adalimumab antibody, or an antigen binding portionthereof.

I. DEFINITIONS

In order that the present invention may be more readily understood,certain terms are first defined.

As used herein, the terms “acidic species”, “acidic region”, and “AR,”refer to the variants of a protein, e.g., an antibody or antigen-bindingportion thereof, which are characterized by an overall acidic charge.For example, in monoclonal antibody (mAb) preparations, such acidicspecies can be detected by various methods, such as ion exchange, forexample, WCX-10 HPLC (a weak cation exchange chromatography), or IEF(isoelectric focusing). Acidic species of an antibody may include chargevariants, structure variants, and/or fragmentation variants. Exemplarycharge variants include, but are not limited to, deamidation variants,afucosylation variants, methylglyoxal (MGO) variants, glycationvariants, and citric acid variants. Exemplary structure variantsinclude, but are not limited to, glycosylation variants and acetonationvariants. Exemplary fragmentation variants include any truncated proteinspecies from the target molecule due to dissociation of peptide chain,enzymatic and/or chemical modifications, including, but not limited to,Fc and Fab fragments, fragments missing a Fab, fragments missing a heavychain variable domain, C-terminal truncation variants, variants withexcision of N-terminal Asp in the light chain, and variants havingN-terminal truncation of the light chain. Other acidic species variantsinclude variants containing unpaired disulfides, host cell proteins, andhost cell nucleic acids, chromatographic materials, and mediacomponents.

In certain embodiments, a protein composition can comprise more than onetype of acidic species variant. For example, but not by way oflimitation, the total acidic species can be divided based onchromatographic retention time of the peaks appearing, for example, in aWCX-10 Weak Cation Exchange HPLC of the protein preparation. FIG. 2depicts a non-limiting example of such a division wherein the totalacidic species associated with the expression of adalimumab is dividedinto a first acidic species region (AR1) and a second acidic speciesregion (AR2).

AR1 can comprise, for example, charge variants such as deamidationvariants, MGO modified species, glycation variants, and citric acidvariants, structural variants such as glycosylation variants andacetonation variants, and/or fragmentation variants. In anotherembodiment, AR2 can comprise, for example, charge variants such asglycation variants and deamidation variants.

With respect, in particular, to adalimumab (and antibodies sharingcertain structural characteristics of adalimumab, e.g., one or more CDRand/or heavy and light chain variable regions of adalimumab), AR1 chargevariants can comprise, but are not limited to, deamidation variants,glycation variants, afucosylation variants, MGO variants or citric acidvariants. In one embodiment, deamidation variants result fromdeamidation occurring at asparagine residues comprising Asn393 andAsn329 and at glutamine residues comprising Gln3 and Gln6. In anotherembodiment, the glycation variants result from glycation occurring atLys98 and Lys151. AR1 structure variants can comprise, but are notlimited to, glycosylation variants or acetonation variants.

AR1 fragmentation variants can comprise Fc and Fab fragments, fragmentsmissing a Fab, fragments missing a heavy chain variable domain,C-terminal truncation variants, variants with excision of N-terminal Aspin the light chain, and variants having N-terminal truncation of thelight chain.

AR2 charge variants can comprise, but are not limited to, deamidationvariants or glycation variants, wherein the deamidation variants canresult from deamidation occurring at asparagine residues comprisingAsn393 and Asn329 and at glutamine residues comprising Gln3 and Gln6,and the glycation variants can result from glycation occurring at Lys98and Lys151.

The term “acidic species” does not include process-related impurities.The term “process-related impurity,” as used herein, refers toimpurities that are present in a composition comprising a protein butare not derived from the protein itself. Process-related impuritiesinclude, but are not limited to, host cell proteins (HCPs), host cellnucleic acids, chromatographic materials, and media components. A “lowprocess-related impurity composition,” as used herein, refers to acomposition comprising reduced levels of process-related impurities ascompared to a composition wherein the impurities were not reduced. Forexample, a low process-related impurity composition may contain about10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less ofprocess-related impurities. In one embodiment, a low process-relatedimpurity composition is free of process-related impurities or issubstantially free of process-related impurities.

The acidic species may be the result of product preparation (referred toherein as “preparation-derived acidic species”), or the result ofstorage (referred to herein as “storage-derived acidic species”).Preparation-derived acidic species are acidic species that are formedduring the preparation (upstream and/or downstream processing) of theprotein, e.g., the antibody or antigen-binding portion thereof. Forexample, preparation-derived acidic species can be formed during cellculture (“cell culture-derived acidic species”). Storage-derived acidicspecies are acidic species that may or may not be present in thepopulation of proteins directly after preparation, but are formed orgenerated while the sample is being stored. The type and amount ofstorage-derived acidic species can vary based on the formulation of thesample. Formation of storage-derived acidic species can be partially orcompletely inhibited when the preparation is stored under particularconditions. For example, an aqueous formulation can be stored at aparticular temperature to partially or completely inhibit AR formation.For example, formation or storage-derived AR can be partially inhibitedin an aqueous formulation stored at between about 2° C. and 8° C., andcompletely inhibited when stored at −80° C. In addition, a low ARcomposition can be lyophilized or freeze-dried to partially orcompletely inhibit the formation of storage-derived AR.

The term “low acidic species composition,” or “low AR composition,” asused herein, refers to a composition comprising an antibody orantigen-binding portion thereof, wherein the composition contains lessthan about 15% acidic species. As used herein, the percent AR in the lowAR composition refers to the weight of the acidic species in a sample inrelation to the weight of the total antibodies contained in the sample.For example, the percent AR can be calculated using weak cation exchangechromatography such as WCX-10, as described herein and also in Example 1of U.S. Provisional Patent Application 61/893,068, entitled “Low AcidicSpecies Compositions and Methods for Producing the Same”, AttorneyDocket Number 117813-73901, filed on Oct. 18, 2013, the entire contentsof which are expressly incorporated herein by reference.

In one embodiment, a low AR composition of the invention may compriseabout 15% or less AR, 14% or less AR, 13% or less AR, 12% or less AR,11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or lessAR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5%or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9% or lessAR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR,1.4% or less AR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1%or less AR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% orless AR, 0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or lessAR, 0.1% or less AR, or 0.0% AR, and ranges within one or more of thepreceding. A low AR composition of the invention may also comprise about0.0% to about 10% AR, about 0.0% to about 5% AR, about 0.0% to about 4%AR, about 0.0% to about 3% AR, about 0.0% to about 2% AR, about 3% toabout 5% AR, about 5% to about 8% AR, or about 8% to about 10% AR, orabout 10% to about 15% AR, and ranges within one or more of thepreceding.

A low AR composition of the invention may comprise about 15% or lessAR1, 14% or less AR1, 13% or less AR1, 12% or less AR1, 11% or less AR1,10% or less AR1, 9% or less AR1, 8% or less AR1, 7% or less AR1, 6% orless AR1, 5% or less AR1, 4.5% or less AR1, 4% or less AR1, 3.5% or lessAR1, 3% or less AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or less AR1,1.8% or less AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or less AR1,1.4% or less AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or less AR1,1% or less AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7% or less AR1,0.6% or less AR1, 0.5% or less AR1, 0.4% or less AR1, 0.3% or less AR1,0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1, and ranges within oneor more of the preceding. A low AR composition of the invention may alsocomprise about 0.0% to about 10% AR1, about 0.0% to about 5% AR1, about0.0% to about 4% AR1, about 0.0% to about 3% AR1, about 0.0% to about 2%AR1, about 3% to about 5% AR1, about 5% to about 8% AR1, or about 8% toabout 10% AR1, or about 10% to about 15% AR1, and ranges within one ormore of the preceding.

A low AR composition of the invention may also comprise about 15% orless AR2, 14% or less AR2, 13% or less AR2, 12% or less AR2, 11% or lessAR2, 10% or less AR2, 9% or less AR2, 8% or less AR2, 7% or less AR2, 6%or less AR2, 5% or less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% orless AR2, 3% or less AR2, 2.5% or less AR2, 2% or less AR2, 1.9% or lessAR2, 1.8% or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or lessAR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or lessAR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or lessAR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% or lessAR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, and ranges withinone or more of the preceding. A low AR composition of the invention mayalso comprise about 0.0% to about 10% AR2, about 0.0% to about 5% AR2,about 0.0% to about 4% AR2, about 0.0% to about 3% AR2, about 0.0% toabout 2% AR2, about 3% to about 5% AR2, about 5% to about 8% AR2, orabout 8% to about 10% AR2, or about 10% to about 15% AR2, and rangeswithin one or more of the preceding.

In one embodiment, a low AR composition comprises between about 0.0% andabout 3% AR1. In another embodiment, a low AR composition comprisesabout between about 0.0% and about 3% AR2. In a preferred embodiment, alow acidic species composition comprises about 3% or less AR2.

In another embodiment, the low AR composition comprises about 1.4% orless AR. For example, in one embodiment, the composition comprises about1.4% AR2 and about 0.0% AR1.

In one embodiment, a low AR composition of the invention may compriseabout 15% or less, 14% or less, 13% or less, 12% or less, 11% or less,10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% orless, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% orless, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% orless, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% orless, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, or 0.0% ofone or more of a deamidation variant, an afucosylation variant, an MGOvariant, a glycation variant, a citric acid variant, a glycosylationvariant, an acetonation variant, or a fragmentation variant, and rangeswithin one or more of the preceding. In one aspect of this embodiment, alow AR composition of the invention may also comprise about 0.0% toabout 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% toabout 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% toabout 8%, or about 8% to about 10%, or about 10% to about 15%, of one ormore of a deamidation variant, an afucosylation variant, an MGO variant,a glycation variant, a citric acid variant, a glycosylation variant, anacetonation variant, or a fragmentation variant, and ranges within oneor more of the preceding. For example, a low AR composition of theinvention may comprise less than 15% of a deamidation variant, whileeach of the other acidic variants, alone or in combination, are at apercentage that is greater than 15%.

The term “non-low acidic species composition,” as used herein, refers toa composition comprising an antibody or antigen-binding portion thereof,which contains more than about 13% acidic species. For example, anon-low acidic species composition may contain about 16% or more, 17% ormore, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more,23% or more, 24% or more, or 25% or more acidic species. In oneembodiment, a non-low acidic species composition can comprise about 5%or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more,11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% ormore, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more,22% or more, 23% or more, 24% or more, or 25% or more of AR1. In anotherembodiment, a non-low acidic species composition can comprise about 10%or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% ormore, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more,21% or more, 22% or more, 23% or more, 24% or more, or 25% or more ofAR2, and ranges within one or more of the preceding.

In one embodiment, a low AR composition has improved biological andfunctional properties, including increased efficacy in the treatment orprevention of a disorder in a subject, e.g., a disorder in which TNFαactivity is detrimental, as compared to a non-low acidic speciescomposition. In one embodiment, the low AR composition comprises ananti-TNFα antibody, or antigen-binding portion thereof, such asadalimumab or a fragment thereof. For example, in one embodiment, a lowAR composition comprising an antibody, or antigen-binding portionthereof, exhibits increased cartilage penetration, decreased boneerosion, and/or reduced cartilage destruction, as compared to a non-lowacidic species composition comprising the same antibody or antigenbinding portion thereof, when administered to a subject suffering from adisorder in which TNFα activity is detrimental.

As used herein, the term “increased cartilage penetration” refers toincreased penetration of cartilage in vivo by a low AR composition ascompared to a non-low AR composition comprising the same antibody orantigen binding portion thereof.

As used herein, the term “reduced cartilage destruction” refers tomeasurable decrease in destruction of cartilage tissue in vivo by a lowAR composition as compared to a non-low AR composition comprising thesame antibody or antigen binding portion thereof. As used herein, theterm “decreased bone erosion” refers to measurable decrease, in vivo, ofthe erosion of bone tissue by a low AR composition as compared to anon-low acidic species composition comprising the same antibody orantigen binding portion thereof. For example, an in vivo model of adisease or disorder in which TNFα activity is detrimental, e.g., a mousemodel of arthritis, can be used to measure cartilage penetration, boneerosion, and/or cartilage destruction by a composition comprising ananti-TNFα antibody or antigen binding portion thereof. One non-limitingexample of an art-recognized mouse model of arthritis is the human TNFtransgenic 197 mouse model of arthritis (TNF-Tg197) (see Keffer, J. etal., EMBO J (1991) 10:4025-4031, the contents of which are expresslyincorporated herein by reference, for further description of theTNF-Tg197 model of arthritis).

In another embodiment, a low AR composition comprising an antibody, orantigen-binding portion thereof, exhibits increased protection againstthe development of arthritis, as measured by arthritic scores, and/orhistopathology scores as compared to a non-low acidic speciescomposition when administered to an animal model of arthritis, e.g., theTNF-Tg197 model of arthritis. As used herein, “arthritic scores” referto signs and symptoms of arthritis in an animal model of arthritis. Asused herein, “histopathology scores” refer to radiologic damageinvolving cartilage and bone as well as local inflammation.

In another embodiment, a low AR composition comprising an antibody, orantigen-binding portion thereof, exhibits reduced synovialproliferation, reduced cell infiltration, reduced chondrocyte death,and/or reduced proteoglycan loss as compared to a non-low acidic speciescomposition. In another embodiment, a low AR composition comprising ananti-TNFα antibody, or antigen-binding portion thereof, exhibitsincreased TNFα affinity as compared to a non-low acidic speciescomposition.

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, or synovial fluid of the subject), which can bedetected, for example, using an anti-TNFα antibody as described above.There are numerous examples of disorders in which TNFα activity isdetrimental. In one embodiment, the disorder in which TNFα activity isdetrimental is an autoimmune disorder. In one embodiment, the autoimmunedisorder is selected from the group consisting of rheumatoid arthritis,juvenile idiopathic arthritis, rheumatoid spondylitis, ankylosingspondylitis, psoriasis, osteoarthritis, gouty arthritis, an allergy,multiple sclerosis, psoriatic arthritis, autoimmune diabetes, autoimmuneuveitis, nephrotic syndrome, juvenile rheumatoid arthritis, Crohn'sdisease and ulcerative colitis. Disorders in which TNFα activity isdetrimental are set forth in U.S. Pat. No. 6,090,382 and also in the“Highlights of HUMIRA® Prescribing Information” for HUMIRA® (adalimumab)Injection (Revised January 2008) the entire contents of which are herebyincorporated herein by reference. The use of TNFα antibodies andantibody portions obtained using methods of the invention for thetreatment of specific disorders is discussed in further detail below.

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., in the case of adalimumab,hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment comprising the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentcomprising the VH and CH1 domains; (iv) a Fv fragment comprising the VLand VH domains of a single arm of an antibody, (v) a dAb fragment (Wardet al., (1989) Nature 341:544-546, the entire teaching of which isincorporated herein by reference), which comprises a VH domain; and (vi)an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv); see, e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883, the entire teachings of which are incorporated herein byreference). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (see,e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody or antigen-binding portion thereof may be part of alarger immunoadhesion molecule, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101, the entire teaching of which is incorporated herein byreference) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058,the entire teaching of which is incorporated herein by reference).Antibody portions, such as Fab and F(ab′)2 fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.In one aspect, the antigen binding portions are complete domains orpairs of complete domains.

The terms “Kabat numbering” “Kabat definitions” and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e., hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, the entire teachings ofwhich are incorporated herein by reference). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), e.g., in the CDRs and in particular CDR3. Themutations can be introduced using the “selective mutagenesis approach.”The human antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In one embodiment, these replacements arewithin the CDR regions. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, theentire teaching of which is incorporated herein by reference) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or back-mutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hTNFα is substantially free ofantibodies that specifically bind antigens other than hTNFα). Anisolated antibody that specifically binds hTNFα may bind TNFα moleculesfrom other species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals. A suitable anti-TNFαantibody is adalimumab.

As used herein, the term “adalimumab,” also known by its trade nameHUMIRA® (AbbVie) refers to a human IgG₁ antibody that binds human tumornecrosis factor α (TNFα). In general, the heavy chain constant domain 2(CH2) of the adalimumab IgG-Fc region is glycosylated through covalentattachment of oligosaccharide at asparagine 297 (Asn-297). The HUMIRA®Prescribing Information, the contents of each of which are expresslyincorporated by reference herein. The light chain variable region ofadalimumab is provided herein as SEQ ID NO:1, and the heavy chainvariable region of adalimumab is provided herein as SEQ ID NO:2. Thelight chain of adalimumab is provided herein as SEQ ID NO:11, and theheavy chain of adalimumab is provided herein as SEQ ID NO:12. Adalimumabcomprises a light chain variable region comprising a CDR1 of SEQ IDNO:7, a CDR2 of SEQ ID NO:5, and a CDR3 of SEQ ID NO:3. Adalimumabcomprises a heavy chain variable region comprising a CDR1 of SEQ IDNO:8, a CDR2 of SEQ ID NO:6 and CDR3 of SEQ ID NO:4. Adalimumab isdescribed in U.S. Pat. Nos. 6,090,382; 6,258,562; 6,509,015; 7,223,394;7,541,031; 7,588,761; 7,863,426; 7,919,264; 8,197,813; 8,206,714;8,216,583; 8,420,081; 8,092,998; 8,093,045; 8,187,836; 8,372,400;8,034,906; 8,436,149; 8,231,876; 8,414,894; 8,372,401, and PCTPublication No. WO2012/065072, the entire contents of each which areexpressly incorporated herein by reference in their entireties.Adalimumab is also described in the “Highlights of PrescribingInformation” for HUMIRA® (adalimumab) Injection (Revised January 2008)the contents of which are hereby incorporated herein by reference.

In one embodiment, adalimumab dissociates from human TNFα with a Kd of1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10-M orless. In another embodiment, adalimumab dissociates from human TNFα witha K_(off) of 5×10⁻⁴ s⁻¹ or less, or with a K_(off) of 1×10⁻⁴ s⁻¹ orless. In still another embodiment, adalimumab neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10⁻⁸ Mor less, an IC50 of 1×10⁻⁹ M or less or an IC50 of 1×10⁻¹° M or less.

Analysis of adalimumab has shown that it has three main basic variants(i.e., Lys 0, Lys 1, and Lys 2), referred to herein as “lysine variantspecies.” As used herein, the term “lysine variant species” refers to anantibody, or antigen-binding portion thereof, comprising heavy chainswith either zero, one or two C-terminal lysines. For example, the “Lys0” variant comprises an antibody, or antigen-binding portion thereof,with heavy chains that do not comprise a C-terminal lysine. The “Lys 1”variant comprises an antibody, or antigen-binding portion thereof, withone heavy chain that comprises a C-terminal lysine. The “Lys 2” variantcomprises an antibody with both heavy chains comprising a C-terminallysine. Lysine variant species can be detected by various methods, suchas ion exchange, for example, by weak cation exchange chromatography(such as WCX-10) of the expression product of a host cell expressing theantibody, or antigen-binding portion thereof. For example, but not byway of limitation, FIG. 2 depicts analysis of adalimumab wherein thethree lysine variants, as well as the two acidic species regions, areresolved from each other.

A composition of the invention may comprise more than one lysine variantspecies of an antibody, or antigen-binding portion thereof. For example,in one embodiment, the composition may comprise a Lys 2 variant of anantibody, or antigen-binding portion thereof. The composition maycomprise a Lys 1 variant of an antibody, or antigen-binding portionthereof. The composition may comprise a Lys 0 variant of an antibody, orantigen-binding portion thereof. In another embodiment, the compositionmay comprise both Lys 1 and Lys 2, or Lys 1 and Lys 0, or Lys 2 and Lys0 variants of an antibody, or antigen-binding portion thereof. Inanother embodiment, the composition may comprise all three lysinevariant species, i.e., Lys 0, Lys 1 and Lys 2, of an antibody, orantigen-binding portion thereof.

In one embodiment, the invention comprises a composition comprising anantibody, or antigen-binding portion thereof, wherein the compositioncomprises less than about 50% lysine variant species that lack aC-terminal lysine (Lys 0). In another embodiment, the compositioncomprises less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%,40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%,26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variantspecies that lack a C-terminal lysine (“Lys 0”). In another embodiment,the composition comprises about 50% to about 0%, about 40% to about 10%,about 30% to about 20%, about 40% to about 20%, or about 30% to about15% lysine variant species that lack a C-terminal lysine (Lys 0). In oneembodiment, the composition comprises 0% lysine variant species thatlack a C-terminal lysine (Lys 0). As used herein, the percent lysinevariant species in the composition refers to the weight of the specificlysine variant species in a sample in relation to the weight of thetotal lysine variant species sum (i.e., the sum of Lys 0, Lys 1 and Lys2) contained in the sample or composition. For example, the percentlysine variant species can be calculated using weak cation exchangechromatography such as WCX-10, as described herein.

In another embodiment, the composition comprises less than about 25%lysine variant species that have one C-terminal lysine (Lys 1). Inanother embodiment, the composition comprises less than about 24%, 23%,22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variant species that have oneC-terminal lysine (Lys 1). In another embodiment, the compositioncomprises about 25% to about 0%, about 20% to about 5%, about 15% toabout 10%, about 20% to about 10%, about 15% to about 5%, or about about25% to about 5% lysine variant species that have one C-terminal lysine(Lys 1). In one embodiment, the composition comprises 0% lysine variantspecies that have one C-terminal Lysine (Lys 1).

In another embodiment, the composition comprises at least about 70%lysine variant species that have two C-terminal lysines (Lys 2). Inanother embodiment, the composition comprises at least about 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%lysine variant species that have two C-terminal lysines (Lys 2). In oneembodiment, the composition comprises about 70% to about 100%, about 70%to about 90%, about 70% to about 80%, about 80% to about 100%, about 85%to about 100%, about 90% to about 100%, about 95% to about 100%, about80% to about 90%, about 85% to about 95%, about 75% to about 85%, orabout 97% to about 100% lysine variant species that have two C-terminallysines (Lys 2). In one embodiment, the composition comprises 100%lysine variant species that have two C-terminal lysines (Lys 2).

As used herein, the term “upstream process technology,” in the contextof protein, e.g., antibody, preparation, refers to activities involvingthe production and collection of proteins (e.g. antibodies) from cells(e.g., during cell culture of a protein of interest). As used herein,the term “cell culture” refers to methods for generating and maintaininga population of host cells capable of producing a recombinant protein ofinterest, as well as the methods and techniques for optimizing theproduction and collection of the protein of interest. For example, oncean expression vector has been incorporated into an appropriate host, thehost can be maintained under conditions suitable for expression of therelevant nucleotide coding sequences, and the collection andpurification of the desired recombinant protein.

When using the cell culture techniques of the instant invention, theprotein of interest can be produced intracellularly, in the periplasmicspace, or directly secreted into the medium. In embodiments where theprotein of interest is produced intracellularly, the particulate debris,either host cells or lysed cells (e.g., resulting from homogenization)can be removed by a variety of means, including but not limited to,centrifugation or ultrafiltration. Where the protein of interest issecreted into the medium, supernatants from such expression systems canbe first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit.

As used herein, the term “downstream process technology” refers to oneor more techniques used after the upstream process technologies topurify the protein, e.g., antibody, of interest. For example, downstreamprocess technology includes purification of the protein product, using,for example, displacement chromatography, affinity chromatography,including Protein A affinity chromatography, ion exchangechromatography, such as anion or cation exchange chromatography,hydrophobic interaction chromatography, or displacement chromatography.

The phrase “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3), e.g., those that bind hTNFα, includes a nucleic acid molecule inwhich the nucleotide sequences encoding the antibody or antibody portionare free of other nucleotide sequences encoding antibodies or antibodyportions that bind antigens other than hTNFα, which other sequences maynaturally flank the nucleic acid in human genomic DNA. Thus, e.g., anisolated nucleic acid of the invention encoding a VH region of ananti-TNFα antibody contains no other sequences encoding other VH regionsthat bind antigens other than, for example, hTNFα. The phrase “isolatednucleic acid molecule” is also intended to include sequences encodingbivalent, bispecific antibodies, such as diabodies in which VH and VLregions contain no other sequences other than the sequences of thediabody.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

As used herein, the term “recombinant protein” refers to a proteinproduced as the result of the transcription and translation of a genecarried on a recombinant expression vector that has been introduced intoa host cell. In certain embodiments the recombinant protein is anantibody, e.g., a chimeric, humanized, or fully human antibody. Incertain embodiments the recombinant protein is an antibody of an isotypeselected from group consisting of: IgG (e.g., IgG1, IgG2, IgG3, IgG4),IgM, IgA1, IgA2, IgD, or IgE. In certain embodiments the antibodymolecule is a full-length antibody (e.g., an IgG1 or IgG4immunoglobulin) or alternatively the antibody can be a fragment (e.g.,an Fc fragment or a Fab fragment).

The term “preparative scale,” as used herein, refers to a scale ofpurification operation that can be readily scaled-up and implemented atlarge scale manufacturing while still providing desired separation. Forinstance, one skilled in the field may develop a process using, e.g., a0.5 cm (i.d.)×20 cm (L) column in the lab, and transfer it to largescale production using, e.g., a 30 cm (i.d.)×20 cm (L) column packedwith the same resin and operated with the same set of buffers, samelinear flow rates (or residence times) and buffer volumes. Inpreparative scale separation, column bed height is typically≦about 30 cmand column pressure drop≦about 5 bar.

The phrase “displacing buffer”, “displacer buffer”, “displacing washbuffer”, or “displacer wash buffer”, as used herein, refers to a bufferthat comprises a displacer molecule. The phrase “displacer molecule”, asused herein, refers to a molecule employed to displace from thechromatographic support components of the mixture to be separated.Selection of a particular displacer molecule will, therefore, bedependent on the chromatographic support employed as well as the proteinsystem. Regardless of which chromatographic support is employed,displacer molecules will generally be selected such that they have ahigh affinity for the support. However, in certain embodiments, adisplacer molecule may be selected that has a reduced affinity for thesupport, so long as it retains the ability to induce a displacementtrain that includes the protein of interest. In certain non-limitingembodiments, the displacer molecule will be employed in the context ofprotein separations in ion exchange chromatography and can be selectedfrom, but not limited to, the group consisting of: polyelectrolytes;polysaccharides; low-molecular-mass dendrimers; amino acids; peptide;antibiotics; and aminoglycosidepolyamines. In certain embodiments thedisplacer is selected from, but not limited to, the group consisting of:Expell SP1™ (for CEX and for mixed mode); Expell Q3 (for anion-exchangechromatography (AEX) and for mixed mode); Propel Q2 (for AEX and formixed mode); and protamine sulfate (for CEX and for mixed mode).Exemplary displacer molecules are described in U.S. Pat. No. 7,632,409,WO 99/47574, WO 03074148, WO2007/055896, WO 2007/064809; and U.S. Pat.No. 6,881,540.

As used herein, the term“aggregates” means agglomeration oroligomerization of two or more individual molecules, including but notlimiting to, protein dimers, trimers, tetramers, oligomers and otherhigh molecular weight species. Protein aggregates can be soluble orinsoluble.

II. LOW ACIDIC SPECIES COMPOSITIONS OF THE INVENTION

The present invention provides low AR compositions comprising a protein,e.g., an antibody, or antigen-binding portion thereof, such asadalimumab, where the composition comprises about 15% or less AR, 14% orless AR, 13% or less AR, 12% or less AR, 11% or less AR, 10% or less AR,9% or less AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or lessAR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR, 2.5%or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% orless AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or lessAR, 1.2% or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR,0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR, 0.4%or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or 0.0%AR, and ranges within one or more of the preceding. A low AR compositionof the invention may also comprise about 0.0% to about 10% AR, about0.0% to about 5% AR, about 0.0% to about 4% AR, about 0.0% to about 3%AR, about 0.0% to about 2% AR, about 3% to about 5% AR, about 5% toabout 8% AR, or about 8% to about 10% AR, or about 10% to about 15% AR,and ranges within one or more of the preceding.

In one embodiment, a low AR composition of the invention may compriseabout 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% or lessAR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1,7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% orless AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or lessAR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or lessAR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or lessAR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or lessAR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or lessAR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1,and ranges within one or more of the preceding. A low AR composition ofthe invention may also comprise about 0.0% to about 10% AR1, about 0.0%to about 5% AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% AR1,about 0.0% to about 2% AR1, about 3% to about 5% AR1, about 5% to about8% AR1, or about 8% to about 10% AR1, or about 10% to about 15% AR1, andranges within one or more of the preceding.

In another embodiment, a low AR composition of the invention may alsocomprise about 15% or less AR2, 14% or less AR2, 13% or less AR2, 12% orless AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% or lessAR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or less AR2,4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2, 2%or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2, 1.6%or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2%or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% orless AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% orless AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0%AR2, and ranges within one or more of the preceding. A low ARcomposition of the invention may also comprise about 0.0% to about 10%AR2, about 0.0% to about 5% AR2, about 0.0% to about 4% AR2, about 0.0%to about 3% AR2, about 0.0% to about 2% AR2, about 3% to about 5% AR2,about 5% to about 8% AR2, or about 8% to about 10% AR2, or about 10% toabout 15% AR2, and ranges within one or more of the preceding.

A low AR composition of the invention may further comprise more than onelysine variant species of an antibody, or antigen-binding portionthereof. For example, in a preferred embodiment, the composition maycomprise a Lys 2 variant of an antibody, or antigen-binding portionthereof. The composition may comprise a Lys 1 variant of an antibody, orantigen-binding portion thereof. The composition may comprise a Lys 0variant of an antibody, or antigen-binding portion thereof. In anotherembodiment, the composition may comprise both Lys 1 and Lys 2, or Lys 1and Lys 0, or Lys 2 and Lys 0 variants of an antibody, orantigen-binding portion thereof. In another embodiment, the compositionmay comprise all three lysine variant species, i.e., Lys 0, Lys 1 andLys 2, of an antibody, or antigen-binding portion thereof.

In one embodiment, a low AR composition of the invention comprises lessthan about 50% lysine variant species that lack a C-terminal lysine (Lys0). In another embodiment, the composition comprises less than about55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%,41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%,27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variantspecies that lack a C-terminal lysine (“Lys 0”). In another embodiment,the composition comprises about 50% to about 0%, about 40% to about 10%,about 30% to about 20%, about 40% to about 20%, or about 30% to about15% lysine variant species that lack a C-terminal lysine (Lys 0). In oneembodiment, the composition comprises 0% lysine variant species thatlack a C-terminal lysine (Lys 0).

In one embodiment, a low AR composition comprises less than about 25%lysine variant species that have one C-terminal lysine (Lys 1). Inanother embodiment, the composition comprises less than about 24%, 23%,22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2% or 1% lysine variant species that have oneC-terminal lysine (Lys 1). In another embodiment, the compositioncomprises about 25% to about 0%, about 20% to about 5%, about 15% toabout 10%, about 20% to about 10%, about 15% to about 5%, or about 25%to about 5% lysine variant species that have one C-terminal lysine (Lys1). In one embodiment, the composition comprises 0% lysine variantspecies that have one C-terminal Lysine (Lys 1).

In another embodiment, a low AR composition of the invention comprisesat least about 70% lysine variant species that have two C-terminallysines (Lys 2). In another embodiment, the composition comprises atleast about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% lysine variant species that have two C-terminal lysines (Lys2). In one embodiment, the composition comprises about 70% to about100%, about 70% to about 90%, about 70% to about 80%, about 80% to about100%, about 85% to about 100%, about 90% to about 100%, about 95% toabout 100%, about 80% to about 90%, about 85% to about 95%, about 75% toabout 85%, or about 97% to about 100% lysine variant species that havetwo C-terminal lysines (Lys 2). In one embodiment, the compositioncomprises 100% lysine variant species that have two C-terminal lysines(Lys 2).

As demonstrated herein, these low AR compositions of the invention haveimproved biological properties. For example, the low AR compositions ofthe invention are characterized by increased cartilage tissuepenetration, reduced cartilage destruction, reduced synovialproliferation, reduced bone erosion, increased protection against thedevelopment of arthritic scores and/or histopathology scores, reducedcell infiltration, reduced proteoglycan loss, reduced chondrocyte death,and/or increased TNFα affinity, as compared to non-low acidic speciescompositions. In addition, the compositions of the present inventionexhibit increased therapeutic efficacy when administered to a subject.

In one embodiment, the protein in the low AR composition of theinvention is an antibody or antigen binding portion thereof. Forexample, the antibody, or antigen binding portion thereof may be ananti-TNFα antibody, or antigen binding portion thereof, such asadalimumab, or an antigen binding portion thereof. In one aspect of thisembodiment, the antibody, or antigen binding portion thereof, cancomprise a light chain variable region comprising the sequence set forthas SEQ ID NO:1, and a heavy chain variable region comprising thesequence set forth as SEQ ID NO:2. In another aspect of this embodiment,the antibody can comprise a light chain variable region comprising aCDR1 having the sequence set forth as SEQ ID NO:7, a CDR2 having thesequence set forth as SEQ ID NO:5, and a CDR3 having the sequence setforth as SEQ ID NO:3. In another aspect of this embodiment, the antibodycan comprise a heavy chain variable region comprising a CDR1 having thesequence set forth as SEQ ID NO:8, a CDR2 having the sequence set forthas SEQ ID NO:6 and a CDR3 having the sequence set forth as SEQ ID NO:4.

The antibody, or antigen binding portion thereof, used in the low ARcompositions of the invention, may be a human, humanized, or chimericantibody.

The antibodies that can be used in the low AR compositions of thepresent disclosure can be generated by a variety of techniques,including immunization of an animal with the antigen of interestfollowed by conventional monoclonal antibody methodologies e.g., thestandard somatic cell hybridization technique of Kohler and Milstein(1975) Nature 256: 495. Somatic cell hybridization procedures can beused. In principle, other techniques for producing monoclonal antibodycan be employed as well, including viral or oncogenic transformation ofB lymphocytes.

One exemplary animal system for preparing hybridomas is the murinesystem. Hybridoma production is a very well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

An antibody used in the low AR compositions of the invention can be ahuman, a chimeric, or a humanized antibody. Chimeric or humanizedantibodies used in the low AR compositions of the invention can beprepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

In one non-limiting embodiment, the antibodies to be used in the low ARcompositions of the invention are human monoclonal antibodies. Suchhuman monoclonal antibodies can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse® (Medarex, Inc.), KMMouse® (Medarex, Inc.), and XenoMouse® (Amgen). The antibodies, orantigen-binding portions thereof, used in the low AR compositions of theinvention can also be produced using the methods described in U.S. Pat.No. 6,090,382, the entire contents of which is expressly incorporatedherein by reference.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseantibodies of the disclosure. For example, mice carrying both a humanheavy chain transchromosome and a human light chain transchromosome,referred to as “TC mice” can be used; such mice are described inTomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (e.g., Kuroiwa et al. (2002) NatureBiotechnology 20:889-894 and PCT application No. WO 2002/092812) and canbe used to raise antibodies of this disclosure.

Recombinant human antibodies to be used in the low AR compositions ofthe invention can be isolated by screening of a recombinantcombinatorial antibody library, e.g., a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fromhuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612, the entireteachings of which are incorporated herein), examples of methods andreagents particularly amenable for use in generating and screeningantibody display libraries can be found in, e.g., Ladner et al. U.S.Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Doweret al. PCT Publication No. WO 91/17271; Winter et al. PCT PublicationNo. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679;Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCTPublication No. WO 92/01047; Garrard et al. PCT Publication No. WO92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibody Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982; the entire teachings of whichare incorporated herein.

Human monoclonal antibodies to be used in the low AR compositions of theinvention can also be prepared using SCID mice into which human immunecells have been reconstituted such that a human antibody response can begenerated upon immunization. Such mice are described in, for example,U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

In certain embodiments, the human antibodies to be used in the low ARcompositions of the invention are anti-TNFα antibodies and antibodyportions thereof, anti-TNFα-related antibodies and antibody portions,and human antibodies and antibody portions with equivalent properties toanti-TNFα antibodies, such as high affinity binding to hTNFα with lowdissociation kinetics and high neutralizing capacity. In one aspect, theinvention provides low AR compositions containing an isolated humanantibody, or an antigen-binding portion thereof, that dissociates fromhTNFα with a Kd of about 1×10⁻⁸ M or less and a Koff rate constant of1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance. Inspecific non-limiting embodiments, an anti-TNFα antibody to be used inthe low AR compositions of the invention competitively inhibits bindingof adalimumab to TNFα under physiological conditions. In one embodiment,the low AR compositions of the invention comprise adalimumab, or anantigen binding fragment thereof.

Antibodies or fragments thereof to be used in the low AR compositions ofthe invention can be altered wherein the constant region of the antibodyis modified to reduce at least one constant region-mediated biologicaleffector function relative to an unmodified antibody. To modify anantibody of the invention such that it exhibits reduced binding to theFc receptor, the immunoglobulin constant region segment of the antibodycan be mutated at particular regions necessary for Fc receptor (FcR)interactions (see, e.g., Canfield and Morrison (1991) J. Exp. Med.173:1483-1491; and Lund et al. (1991) J. of Immunol. 147:2657-2662, theentire teachings of which are incorporated herein). Reduction in FcRbinding ability of the antibody may also reduce other effector functionswhich rely on FcR interactions, such as opsonization and phagocytosisand antigen-dependent cellular cytotoxicity.

To express an antibody or antigen-binding fragment thereof to be used inthe low AR compositions of the invention, DNAs encoding the protein,such as DNAs encoding partial or full-length light and heavy chains inthe case of antibodies, are inserted into one or more expression vectorsuch that the genes are operatively linked to transcriptional andtranslational control sequences. (See, e.g., U.S. Pat. No. 6,090,382,the entire teaching of which is incorporated herein by reference.) Inthis context, the term “operatively linked” is intended to mean that agene encoding the protein of interest is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the gene. The expression vector and expression controlsequences are chosen to be compatible with the expression host cellused. In certain embodiments, the protein of interest will comprisingmultiple polypeptides, such as the heavy and light chains of anantibody. Thus, in certain embodiments, genes encoding multiplepolypeptides, such as antibody light chain genes and antibody heavychain genes, can be inserted into a separate vector or, more typically,the genes are inserted into the same expression vector. Genes areinserted into expression vectors by standard methods (e.g., ligation ofcomplementary restriction sites on the gene fragment and vector, orblunt end ligation if no restriction sites are present). Prior toinsertion of the gene or genes, the expression vector may already carryadditional polypeptide sequences, such as, but not limited to, antibodyconstant region sequences. For example, one approach to converting theanti-TNFα antibody or anti-TNFα antibody-related VH and VL sequences tofull-length antibody genes is to insert them into expression vectorsalready encoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to the CHsegment(s) within the vector and the VL segment is operatively linked tothe CL segment within the vector. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the protein from a host cell. The gene can becloned into the vector such that the signal peptide is linked in-frameto the amino terminus of the gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to protein coding genes, a recombinant expression vector cancarry one or more regulatory sequence that controls the expression ofthe protein coding genes in a host cell. The term “regulatory sequence”is intended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the protein coding genes. Such regulatory sequencesare described, e.g., in Goeddel; Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990), the entireteaching of which is incorporated herein by reference. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Suitable regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma. For further description of viralregulatory elements, and sequences thereof, see, e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.Pat. No. 4,968,615 by Schaffner et al., the entire teachings of whichare incorporated herein by reference.

A recombinant expression vector may also carry one or more additionalsequences, such as a sequence that regulates replication of the vectorin host cells (e.g., origins of replication) and/or a selectable markergene. The selectable marker gene facilitates selection of host cellsinto which the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al., the entireteachings of which are incorporated herein by reference). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr- host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

An antibody, or antibody portion, to be used in the low AR compositionsof the invention can be prepared by recombinant expression ofimmunoglobulin light and heavy chain genes in a host cell. To express anantibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and secreted intothe medium in which the host cells are cultured, from which medium theantibodies can be recovered. Standard recombinant DNA methodologies areused to obtain antibody heavy and light chain genes, incorporate thesegenes into recombinant expression vectors and introduce the vectors intohost cells, such as those described in Sambrook, Fritsch and Maniatis(eds), Molecular Cloning; A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.Nos. 4,816,397 & 6,914,128, the entire teachings of which areincorporated herein.

For expression of protein, for example, the light and heavy chains of anantibody, the expression vector(s) encoding the protein is (are)transfected into a host cell by standard techniques. The various formsof the term “transfection” are intended to encompass a wide variety oftechniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is theoretically possible to express the proteins of theinvention in either prokaryotic or eukaryotic host cells, expression ofantibodies in eukaryotic cells, such as mammalian host cells, issuitable because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active protein. Prokaryoticexpression of protein genes has been reported to be ineffective forproduction of high yields of active protein (Boss and Wood (1985)Immunology Today 6:12-13, the entire teaching of which is incorporatedherein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated proteins, forexample, glycosylated antibodies, are derived from multicellularorganisms. Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori have beenidentified. A variety of viral strains for transfection are publiclyavailable, e.g., the L-1 variant of Autographa californica NPV and theBm-5 strain of Bombyx mori NPV, and such viruses may be used as thevirus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Mammalian cells can be used for expression and production of therecombinant protein used in the low AR compositions of the invention,however other eukaryotic cell types can also be employed in the contextof the instant invention. See, e.g., Winnacker, From Genes to Clones,VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells forexpressing recombinant proteins according to the invention includeChinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, describedin Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp (1982) Mol.Biol. 159:601-621, the entire teachings of which are incorporated hereinby reference), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding protein genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or secretion of the antibody into theculture medium in which the host cells are grown. Other examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2), the entire teachings of which areincorporated herein by reference.

Host cells are transformed with the above-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce a protein may be cultured in a variety ofmedia. Commercially available media such as Ham's F10™ (Sigma), MinimalEssential Medium™ (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium™ (DMEM), (Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells, the entire teachings of which areincorporated herein by reference. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asgentamycin drug), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

Host cells can also be used to produce portions of intact proteins, forexample, antibodies, including Fab fragments or scFv molecules. It isunderstood that variations on the above procedure are within the scopeof the present invention. For example, in certain embodiments it may bedesirable to transfect a host cell with DNA encoding either the lightchain or the heavy chain (but not both) of an antibody. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to an antigen. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the target antibody,depending on the specificity of the antibody of the invention, bycrosslinking an antibody of the invention to a second antibody bystandard chemical crosslinking methods.

In a suitable system for recombinant expression of a protein, forexample, an antibody, or antigen-binding portion thereof, a recombinantexpression vector encoding the protein, for example, both an antibodyheavy chain and an antibody light chain, is introduced into dhfr-CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the protein gene(s) are each operatively linked toCMV enhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the gene(s). The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the protein, for example, theantibody heavy and light chains, and intact protein, for example, anantibody, is recovered from the culture medium. Standard molecularbiology techniques are used to prepare the recombinant expressionvector, transfect the host cells, select for transformants, culture thehost cells and recover the protein from the culture medium.

When using recombinant techniques, the protein, for example, antibodiesor antigen binding fragments thereof, can be produced intracellularly,in the periplasmic space, or directly secreted into the medium. In oneaspect, if the protein is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed cells (e.g., resultingfrom homogenization), can be removed, e.g., by centrifugation orultrafiltration. Where the protein is secreted into the medium,supernatants from such expression systems can be first concentratedusing a commercially available protein concentration filter, e.g., anAmicon™ or Millipore Pellicon™ ultrafiltration unit.

Some antibodies can be secreted directly from the cell into thesurrounding growth media; others are made intracellularly. Forantibodies made intracellularly, the first step of a purificationprocess typically involves: lysis of the cell, which can be done by avariety of methods, including mechanical shear, osmotic shock, orenzymatic treatments. Such disruption releases the entire contents ofthe cell into the homogenate, and in addition produces subcellularfragments that are difficult to remove due to their small size. Theseare generally removed by differential centrifugation or by filtration.Where the antibody is secreted, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

III. PREPARATION OF LOW AR COMPOSITIONS USING DISPLACEMENTCHROMATOGRAPHY

In certain embodiments, the low AR compositions of the present inventionmay be produced using downstream process technologies (e.g.,displacement chromatography), following cell culture of a protein.

The methods described herein for the production of compositionscomprising low AR and/or low process-related impurities comprise thepurification of a protein, such as an antibody or antigen-bindingportion thereof, using displacement chromatography.

The methods described herein for the production of compositionscomprising low AR and/or low process-related impurities also comprisethe purification of a protein, such as an antibody or antigen-bindingportion thereof, using displacement chromatography in combination with,for example, other types of chromatography, such as multimodal (MM)chromatography, wherein the MM media comprises both ion exchange andhydrophobic interaction functional groups, and an aqueous salt solution.In one embodiment, the same or substantially the same aqueous saltsolution is used as a loading buffer and a wash buffer.

In further embodiments, the methods described herein for the productionof compositions comprising low AR and/or low process-related impuritiescomprise the purification of a protein, such as an antibody orantigen-binding portion thereof, using displacement chromatography incombination with chromatography comprising an anion exchange (AEX) resinand an aqueous salt solution. In one embodiment, the same orsubstantially the same aqueous salt solution is used as a loading bufferand a wash buffer.

In yet further embodiments, the methods described herein for theproduction of compositions comprising low AR and/or low process-relatedimpurities comprise the purification of a protein, such as an antibodyor antigen-binding portion thereof, using displacement chromatography incombination with chromatography comprising a cation exchange (CEX)adsorbent resin and an aqueous salt solution. In one embodiment, thesame or substantially the same aqueous salt solution is used as aloading buffer and a wash buffer, and the target protein bound to theCEX adsorbent resin is eluted with a buffer having a higher conductivityand/or pH than the loading/wash buffer.

In still further embodiments, the methods described herein forproduction of compositions comprising low AR and/or low process-relatedimpurities comprise the purification of a protein, such as an antibodyor antigen-binding portion thereof, using displacement chromatography incombination with several media, for example by using an anion exchange(AEX) resin, and chromatography using a cation exchange (CEX) adsorbentresin, in a suitable buffer, such as, for example, a Tris/Formate buffersystem. In one embodiment, the sample is purified affinitychromatography media, e.g., Protein A, prior to the ion chromatographyresins. For example, in one embodiment, the methods described herein forproduction of compositions comprising low AR comprise the exemplaryprocess reflected in FIG. 190 of U.S. Provisional Application No.61/893,068, entitled “Low Acidic Species Compositions and Methods forProducing and Using the Same”, filed on Oct. 18, 2013, the entirecontents of which are expressly incorporated herein by reference.

In one embodiment, the method for producing a low AR compositioncomprising an antibody, or antigen binding portion thereof, comprisescontacting a first sample comprising the antibody, or antigen bindingportion thereof, to affinity chromatography media in a load buffer (forexample a low concentration Tris/Formate buffer), and eluting the samplefrom the affinity chromatography media as a first eluted sample,contacting the first eluted sample to a first chromatography media, suchas an AEX chromatography resin, in a load buffer, and eluting the samplefrom the AEX chromatography resin as a second eluted sample. The secondeluted sample is then contacted with a second chromatography media, suchas a CEX chromatography resin, in a load buffer, and the sample iseluted from the CEX chromatography resin as a third eluted sample. Inone embodiment, the CEX chromatography resin is eluted one, two, threeor more times. In one embodiment, the process optionally includes one ormore intermediate filtration steps, pH adjustment steps and inactivationsteps.

In one embodiment, the displacement chromatography steps, alone or incombination with other purification steps, produce a low AR compositioncomprising an antibody, or antigen binding portion thereof, whichcontains 15% or less AR, 14% or less AR, 13% or less AR, 12% or less AR,11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or lessAR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5%or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9% or lessAR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR,1.4% or less AR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1%or less AR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% orless AR, 0.5% or less AR, 0.4% AR, 0.3% or less AR, 0.2% or less AR,0.1% or less AR, or 0.0% AR, and ranges within one or more of thepreceding. In one aspect of this embodiment, the low AR composition ofthe invention comprises about 0.0% to about 10% AR, about 0.0% to about5% AR, about 0.0% to about 4% AR, about 0.0% to about 3% AR, about 0.0%to about 2% AR, about 3% to about 5% AR, about 5% to about 8% AR, orabout 8% to about 10% AR, or about 10% to about 15% AR, and rangeswithin one or more of the preceding.

In one embodiment, the displacement chromatography steps, alone or incombination with other purification steps, produce a low AR compositioncomprising an antibody, or antigen binding portion thereof, whichcontains 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% or lessAR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1,7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% orless AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or lessAR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or lessAR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or lessAR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or lessAR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or lessAR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1,and ranges within one or more of the preceding. In one aspect of thisembodiment, the low AR composition of the invention comprises about 0.0%to about 10% AR1, about 0.0% to about 5% AR1, about 0.0% to about 4%AR1, about 0.0% to about 3% AR1, about 0.0% to about 2% AR1, about 3% toabout 5% AR1, about 5% to about 8% AR1, or about 8% to about 10% AR1, orabout 10% to about 15% AR1, and ranges within one or more of thepreceding.

In one embodiment, the displacement chromatography steps, alone or incombination with other purification steps, produce a low AR compositioncomprising an antibody, or antigen binding portion thereof, whichcontains 15% or less AR2, 14% or less AR2, 13% or less AR2, 12% or lessAR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% or less AR2,7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or less AR2, 4% orless AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2, 2% or lessAR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2, 1.6% or lessAR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or lessAR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% or lessAR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or lessAR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2,and ranges within one or more of the preceding. In one aspect of thisembodiment, the low AR composition of the invention comprises about 0.0%to about 10% AR2, about 0.0% to about 5% AR2, about 0.0% to about 4%AR2, about 0.0% to about 3% AR2, about 0.0% to about 2% AR2, about 3% toabout 5% AR2, about 5% to about 8% AR2, or about 8% to about 10% AR2, orabout 10% to about 15% AR2, and ranges within one or more of thepreceding.

Protein Purification Generally

Following upstream processing of a protein of interest, downstreamprocess technologies can be used to purify the protein. For example, butnot by way of limitation, once a clarified solution or mixturecomprising the protein of interest, for example, an antibody or antigenbinding fragment thereof, has been obtained, separation of the proteinof interest from the process-related impurities, aggregates, fragments,and/or charge variant species (e.g., acidic species and basic species)can be effected using displacement chromatography, either alone or incombination with different purification techniques, including, but notlimited to, affinity separation steps, ion exchange separation steps,mixed mode separation steps, and hydrophobic interaction separationsteps, singularly or in combination. The separation steps separatemixtures of proteins on the basis of their charge, degree ofhydrophobicity, or size, or any combination thereof, depending on theparticular form of separation, including chromatographic separation. Inone aspect of the invention, separation is performed usingchromatography, including cationic, anionic, and hydrophobicinteraction. Several different chromatography resins are available foreach of these techniques, allowing accurate tailoring of thepurification scheme to the particular protein involved. Each of theseparation methods result in the protein traversing at different ratesthrough a column, to achieve a physical separation that increases asthey pass further through the column, or adhere selectively to theseparation medium. The proteins are then differentially eluted bydifferent solvents. In some cases, the antibody is separated fromimpurities when the impurities preferentially adhere to the column andthe antibody less so, i.e., the desired antibody variant is present inthe Flow Through.

In certain embodiments, a low AR composition is produced usingdisplacement chromatography to identify the particular conditions, e.g.,displacers, displacer concentration, salt concentration, pH,temperature, load amount and conditions, and washing conditions,sufficient to elicit the desired fractionation profile, e.g.,fractionation of acidic species and lysine variants, of a samplecomprising the protein of interest and at least one process-relatedimpurity. In certain embodiments, the method further comprises poolingthe resulting fractions comprising the desired low AR composition.

The purification process may begin at the separation step after theantibody has been produced using upstream production methods describedherein and in U.S. Provisional Application No. 61/893,068, entitled “LowAcidic Species Compositions and Methods for Producing and Using theSame”, filed on Oct. 18, 2013, the entire contents of which areincorporated herein by reference, and/or by alternative productionmethods conventional in the art. Once a clarified solution or mixturecomprising the protein of interest, e.g., an antibody, has beenobtained, separation of the protein of interest from process-relatedimpurities, such as the other proteins produced by the cell, as well asproduct-related substances, such as acidic or basic variants, isperformed.

In certain non-limiting embodiments, such separation is performed usingcation exchange (CEX), anion exchange (AEX), and/or mixed mode (MM)chromatography. In certain embodiments, a combination of one or moredifferent purification techniques, including affinity separationstep(s), ion exchange separation step(s), mixed-mode step(s), and/orhydrophobic interaction separation step(s) can also be employed. Suchadditional purification steps separate mixtures of proteins on the basisof their charge, degree of hydrophobicity, and/or size. In one aspect ofthe invention, such additional separation steps are performed usingchromatography, including hydrophobic, anionic or cationic interaction(or a combination thereof). Numerous chromatography resins arecommercially available for each of these techniques, allowing accuratetailoring of the purification scheme to the particular protein involved.Each of the separation methods allow proteins to either traverse atdifferent rates through a column, achieving a physical separation thatincreases as they pass further through the column, or to adhereselectively to a separation resin (or medium). The proteins are thendifferentially eluted using different eluents. In some cases, theprotein of interest is separated from impurities when the impuritiesspecifically adhere to the column's resin and the protein of interestdoes not, i.e., the protein of interest is contained in the effluent,while in other cases the protein of interest will adhere to the column'sresin, while the impurities and/or product-related substances areextruded from the column's resin during a wash cycle.

Primary Recovery and Virus Inactivation

In certain embodiments, the initial steps of the purification methods ofthe present invention involve the clarification and primary recovery ofantibody from a sample matrix. In certain embodiments, the primaryrecovery will include one or more centrifugation steps to separate theantibody product from the cells and cell debris. Centrifugation of thesample can be performed at, for example, but not by way of limitation,7,000×g to approximately 12,750×g. In the context of large scalepurification, such centrifugation can occur on-line with a flow rate setto achieve, for example, but not by way of limitation, a turbidity levelof 150 NTU in the resulting supernatant. Such supernatant can then becollected for further purification, or in-line filtered through one ormore depth filters for further clarification of the sample.

In certain embodiments, the primary recovery will include the use of oneor more depth filtration steps to clarify the sample matrix and therebyaid in purifying the antibodies of interest in the present invention. Inother embodiments, the primary recovery will include the use of one ormore depth filtration steps post centrifugation to further clarify thesample matrix. Non-limiting examples of depth filters that can be usedin the context of the instant invention include the Millistak+X0HC,F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), Cuno™ model30/60ZA, 60/90 ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2μm filter such as Sartorius's 0.45/0.2 μm Sartopore™ bi-layer orMillipore's Express SHR or SHC filter cartridges typically follows thedepth filters.

In certain embodiments, the primary recovery process can also be a pointat which to reduce or inactivate viruses that can be present in thesample matrix. For example, any one or more of a variety of methods ofviral reduction/inactivation can be used during the primary recoveryphase of purification including heat inactivation (pasteurization), pHinactivation, buffer/detergent treatment, UV and γ-ray irradiation andthe addition of certain chemical inactivating agents such asβ-propiolactone or e.g., copper phenanthroline as described in U.S. Pat.No. 4,534,972. In certain embodiments of the present invention, thesample matrix is exposed to detergent viral inactivation during theprimary recovery phase. In other embodiments, the sample matrix may beexposed to low pH inactivation during the primary recovery phase.

In those embodiments where viral reduction/inactivation is employed, thesample mixture can be adjusted, as needed, for further purificationsteps. For example, following low pH viral inactivation, the pH of thesample mixture is typically adjusted to a more neutral pH, e.g., fromabout 4.5 to about 8.5, prior to continuing the purification process.Additionally, the mixture may be diluted with water for injection (WFI)to obtain a desired conductivity.

Affinity Chromatography

In certain embodiments, it will be advantageous to subject a sampleproduced by the techniques of the instant invention to affinitychromatography to further purify the protein of interest away fromacidic species before a displacement chromatography step. In certainembodiments the chromatographic material is capable of selectively orspecifically binding to the protein of interest (“capture”).Non-limiting examples of such chromatographic material include: ProteinA, Protein G, chromatographic material comprising, for example, anantigen bound by an antibody of interest, and chromatographic materialcomprising an Fc binding protein. In specific embodiments, the affinitychromatography step involves subjecting the primary recovery sample to acolumn comprising a suitable Protein A resin. In certain embodiments,Protein A resin is useful for affinity purification and isolation of avariety of antibody isotypes, particularly IgG1, IgG2, and IgG4. ProteinA is a bacterial cell wall protein that binds to mammalian IgGsprimarily through their Fc regions. In its native state, Protein A hasfive IgG binding domains as well as other domains of unknown function.

There are several commercial sources for Protein A resin. One suitableresin is MabSelect™ from GE Healthcare. Suitable resins include, but notlimited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelectXtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra,and ProSep Ultra Plus from EMD Millipore, MapCapture from LifeTechnologies. A non-limiting example of a suitable column packed withMabSelect™ is an about 1.0 cm diameter×about 21.6 cm long column (˜17 mLbed volume). This size column can be used for small scale purificationsand can be compared with other columns used for scale ups. For example,a 20 cm×21 cm column whose bed volume is about 6.6 L can be used forlarger purifications. Regardless of the column, the column can be packedusing a suitable resin such as MabSelect™

Protein A Affinity Chromatography

In certain embodiments, particularly where the protein of interest is anantibody, the composition, e.g., a primary recovery sample, is subjectedto Protein A affinity chromatography before displacement chromatographyto produce the low AR compositions of the invention. There are a varietyof commercial sources for Protein A resin. Suitable resins include, butnot limited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelectXtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra,and ProSep Ultra Plus from EMD Millipore, MapCapture from LifeTechnologies.

The Protein A column can be equilibrated with a suitable buffer prior tosample loading. Following the loading of the column, the column can bewashed one or multiple times using a suitable sets of buffers. TheProtein A column can then be eluted using an appropriate elution buffer.For example, glycine-HCL or citric acid can be used as an elutionbuffer. The eluate can be monitored using techniques well known to thoseskilled in the art. The eluate fractions of interest can be collectedand then prepared for further processing.

The Protein A eluate may subject to a viral inactivation step either bydetergent or low pH, provided this step is not performed prior to theProtein A capture operation. A proper detergent concentration or pH andtime can be selected to obtain desired viral inactivation results. Afterviral inactivation, the Protein A eluate is usually pH and/orconductivity adjusted for subsequent purification steps.

The Protein A eluate may be subjected to filtration through a depthfilter to remove turbidity and/or various impurities from the antibodyof interest prior to additional chromatographic polishing steps.Examples of depth filters include, but not limited to, Millistak+X0HC,F0HC, D0HC, A1HC, and B1HC Pod filters (EMD Millipore), or Zeta Plus30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VR05 filters (3M). The ProteinA eluate pool may need to be conditioned to proper pH and conductivityto obtain desired impurity removal and product recovery from the depthfiltration step.

The invention is not limited to capture of the protein of interest usingProtein A chromatography. A non-Protein A chromatography capture stepcan also be carried out. For example, cation exchange capture andnon-chromatographic methods, such as aqueous two phase extraction orprecipitation, or other methods known in the art, can be used,

Displacement Chromatography

In certain embodiments of the present invention, a protein sample, e.g.,a primary recovery sample from cell culture, a Protein A eluate sample,or a sample having undergone one or more of the other purificationstrategies outlined herein, is subjected to displacement chromatographyto produce the low AR compositions of the invention. In certainembodiments, the displacer molecule is selected to have a higheraffinity for the stationary phase (i.e., the chromatographic support)than the components present in the material to be separated. In certainembodiments, the displacer induces the components of the mixture todevelop into consecutive zones of concentrated and purified species inthe order of decreasing binding affinity ahead of the displacer front (a“displacement train”). In certain embodiments, the displacement processallows for higher column loading levels (as compared to conventionalhigh-resolution chromatographic separations such as bind and lineargradient elution mode) without compromising the purity and recovery ofthe component of interest. In certain embodiments, washing of thedisplacement train from the column using the displacer solution allowsfor the component of interest to be isolated by collecting (and poolingif necessary) the proper fraction(s) of the displaced eluate. Along withacidic species, other product-related substances, such as basic species,product aggregates, and/or product fragments, and process-relatedimpurities, such as HCPs, can be selectively collected or reduced usingdisplacement chromatography.

Displacement chromatography in described, in general, in Brgles et al.,Journal of Chromatography A, 1218 (2011) 2389-2395; Gajdosik et al.,Journal of Chromatography A, 1239 (2012) 1-9; Gerstner et al.,Biotechnol. Prog., (1992), 8, 540-545; Kundu et al., AnalyticalBiochemistry, 248, 111-116, (1997); and Vogt et al., Journal ofChromatography A, 760 (1997) 125-137.

In certain embodiments, the displacer will be employed in the context ofan ion exchange, e.g., anion exchange or cation exchange, or mixed modechromatographic separation. A detailed description of ion exchangechromatography and a listing of exemplary chromatographic supports whichcan be employed in the context of displacement chromatography arepresented below. A detailed description of mixed mode chromatography anda listing of exemplary chromatographic supports which can be employed inthe context of displacement chromatography are also presented below. Incertain non-limiting embodiments, a cation exchange, an anion exchange,or a mixed mode displacement chromatography step is employed toeffectively reduce product-related substances (e.g., acidic speciesand/or basic species such as lysine variant species) from, e.g., amonoclonal antibody feed stream. In specific embodiments, conventional(or relatively weak) binding conditions can be employed and cationicmolecules having high affinity for a CEX, AEX, or multimodal ligand(such as Expell SP1™ and protamine sulfate) can be employed to inducethe formation of a product-related substances displacement train. Incertain of such embodiments, the acidic species variant population isenriched in the front followed by the main isoform, and, thereafter, thebasic population. Thus, in certain embodiments, exclusion of thoseearlier fractions from the remainder eluate results in an AR-reducedproduct. Alternatively, exclusion of the fractions following the mainisoform results in a lysine species variant-reduced product. In certainembodiments, the fragments and aggregates are reduced in an AR-reducedproduct. In certain embodiments, the HCPs are reduced in an AR-reducedproduct.

In certain embodiments, the displacer concentration will be selectedfrom the range of about 0.1 mM to about 10 mM, or about 0.25 mM to about10 mM. In certain embodiments, the displacer concentration will beselected from a range of about 0.1 mM to about 5 mM, or about 0.25 mM toabout 3 mM. In certain embodiments, the displacer concentration will beselected from a range of about 0.1 mM to about 5 mM, or about 0.25 mM toabout 2 mM. In certain embodiments, the displacer concentration will beselected from a range of about 0.1 mM to about 2 mM, or about 0.25 mM toabout 1 mM. In certain embodiments, the displacer concentration will beselected from a concentration of about 0.1 mM to about 1 mM, or about0.25 mM to about 0.5 mM.

In certain embodiments the displacer is Expell SP1™ and the displacerconcentration will be selected from the range of about 0.1 mM to about10 mM, or about 0.25 mM to about 10 mM. In certain embodiments, thedisplacer is protamine sulfate and the displacer concentration will beselected from the range of about 0.1 mM to about 5 mM, or about 0.25 mMto about 5 mM.

In certain embodiments, a displacing buffer is used in one-stepdisplacement process. In certain embodiments, the total volume of theone-step displacing buffer is in the range of about 20 CVs to about 50CVs, or about 25 CVs to about 40 CVs, or about 30 CVs. In anotherembodiment, the total volume of the one-step displacing buffer is about15 CVs, about 20 CVs, about 25 CVs, about 30 CVs, about 35 CVs, about 40CVs, about 45 CVs, or about 50 CVs.

Although displacement chromatography conventionally employs a displacerat a fixed concentration to achieve component separation, an improvedmethod using multiple displacing buffers is also disclosed herein. Forexample, a two-step displacement method is employed where a firstdisplacer concentration is employed for a certain initial number ofcolumn volumes (CVs) and a second, higher, displacer concentration isemployed for a subsequent number of CVs. The total volume of thedisplacing buffers needed to complete the displacement process issignificantly (e.g., 25-45%) less than that needed when using onedisplacing buffer in the one-step displacement process in order toachieve comparable separation performances. In certain embodiments, thefirst displacer concentration is about 0.25 mM, about 0.3 mM, about 0.35mM, about 0.4 mM, about 0.45 mM, or about 0.5 mM. In certainembodiments, the second displacer concentration is about 0.5 mM, about 1mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM,about 4 mM, about 4.5 mM, or about 5 mM.

In certain embodiments, a two-step displacement method is employed wherethe first displacer concentration is employed for up to about 10 CVs. Incertain embodiments, the first displacer concentration is employed forup to about 25 CVs. In another embodiments, the first displacerconcentration is employed for up to about 10, 11 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24 or 25 CVs.

In certain embodiments, the second displacer concentration is employedfor up to about 10 CVs. In certain embodiments, the second displacerconcentration is employed for up to about 25 CVs. In another embodiment,the second displacer concentration is employed for up to about 10, 1112, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 CVs.

In certain embodiments, the total required displacing buffer volume isabout 13 CVs for a two-step displacement process. In certainembodiments, the total required displacer buffer volume is about 15 CVsfor a two-step displacement process. In certain embodiments, the totalrequired displacer buffer volume is about 33 CVs for a two-stepdisplacement process. In another embodiment, the total requireddisplacing buffer volune is about 10, 11, 12, 13, 14, 115, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 CVsfor a two-step displacement process.

One of skill in the art would understand that further reduction inrequired buffer volumes for each displacement step is expected. Incertain embodiments, multiple steps of increasing displacerconcentration are employed. As outlined in the Examples section, below,incorporation of additional displacement concentration steps into thepurification strategy can allow for unexpectedly efficient chargevariant, product aggregate, product fragment, and/or HCP clearance. Forexample, in one embodiment, three, four, five, six, seven, eight, nineor ten displacement concentration steps are used.

In certain embodiments, a linear gradient displacement method isemployed where an initial, low, displacer concentration is followed bythe addition of displacer at increasing concentrations in accordancewith a linear gradient. For example, but not by way of limitation, thedisplacer concentration can range from about 0 mM to about 1 mM over thecourse of about 40 CVs. Again, as outlined in the Examples section,below, incorporation of a linear displacer concentration gradient intothe purification strategy can allow for unexpectedly efficient chargevariant, product aggregate, product fragment, and/or HCP clearance.

In certain embodiments, a displacement buffer consisting of two or moredisplacers is used. In certain embodiments, different displacers areused in the multi-step displacement process. For example, a displacementbuffer consisting of two, three, four, five, six, seven, eight, nine orten displacers may be used. Alternatively, two, three, four five, six,seven, eight, nine or ten displacers may be used in a multi-stepdisplacement process.

In certain embodiments of the present invention, the pH of thedisplacing wash buffer is below the pI of the protein of interest. Incertain embodiments, the pH of the displacing wash buffer is in therange of about 5.0 to about 9.0, about 6.0 to about 8.0, about 7.0 toabout 7.7, or about 7.5 to about 7.7. In another embodiment, the pH ofthe displacing wash buffer is about 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4,8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

In certain embodiments of the present invention, the conductivity of thedisplacing wash buffer is between about 1 to about 86 mS/cm, about 2 toabout 20 mS/cm, about 2 to about 7 mS/cm, or about 5 to about 6.6 mS/cm.In another embodiment, the conductivity of the displacing wash buffer isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 86 mS/cm.

In certain embodiments of the present invention, the column bed heightis between about 10 cm to about 30 cm, about 15 cm to about 25 cm, about20 cm to about 30 cm, or about 25 cm. In another embodiment, the columnbed height is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 cm.

In certain embodiments of the present invention, the flow residence timeis between about 2 minutes to about 25 minutes, about 5 minutes to about20 minutes, about 10 minutes to about 20 minutes, or about 15 minutes toabout 20 minutes. In another embodiment, the flow residence time isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24 or 25 minutes.

In certain embodiments, the displacer buffer pH and displacerconcentration can affect the displacement profile and, as a result,impact clearance of process-related impurities and/or product-relatedsubstances, such as charge variants, in unexpected ways. Thus, effectiveoperating regimes with regard to the reduction of process-relatedimpurities and/or product-related substances depend on the specificprotein-resin-displacer system. For example, but not by way oflimitation, when a feed stream containing Adalimumab is separated usingdisplacement chromatography, significant AR reduction (AAR %) can beachieved using a displacing buffer with pH in the range of 6-8 withdisplacer concentration as low as 0.25-0.5 mM. In fact, as described inExample 2 below, the extent of Adalimumab AR reduction increasessignificantly as pH varies from 6.5 to 7.5, for example, over a 6%decrease in AR level can be achieved at pH 7.5 with a product yield˜75%. In certain embodiments, as outlined in the Examples presentedbelow, the total AR level (%) in Adalimumab product pool can be reducedby over 10% with an acceptable processing yield (≧75%) from a CEXdisplacement chromatography process, or 4-7% from a mixed modedisplacement chromatography process. Similarly, for mAb X, FIG. 32indicates that ΔAR % surprisingly increases from 3.3 to 6.5% as pHvaries from 7 to 7.7 in a mixed mode displacement chromatographyprocess.

In certain embodiments, conditions selected for reducing AR are alsocapable of reducing process-related impurities and/or otherproduct-related substances. For example, but not by way of limitation,conditions selected for AR reduction are also capable of reducingprocess-related impurities, such as HCPs. In additional, non-limitingexamples, conditions selected for AR reduction are also capable ofreducing product-related impurities, such as aggregates and/orfragments.

In certain embodiments, displacement chromatography can be used as thesole method of purification of the protein of interest. In certainembodiments, displacement chromatography can be used in combination withother purification strategies, such as, but not limited to, thealternative purification techniques described herein, to reduceprocess-related impurities and/or other product-related substances. Inone embodiment, displacement chromatography is used following Protein Aaffinity steps.

In certain embodiments, fractions are collected during the displacementstep and are combined (pooled) after appropriate analysis to provide aprotein preparation, which is also referred to herein as a purified orpartially-purified sample, that contains a desired level of the proteinof interest and which can include one or more process-related impuritiesand/or other product-related substances. In certain embodiments, one ormore process monitoring tools can be used in connection with thetechniques described herein to facilitate the identification of aneffective product pooling strategy. In certain embodiments, suchmonitoring can include on-line or in-line process monitoring. Forexample, but not by way of limitation, spectroscopy methods such as UV,NIR, FTIR, Fluorescence, and Raman may be used to monitor levels ofproduct-related species, e.g., acidic species and lysine variants, in anon-line, at line or in-line mode. These methods allow for the productionof data that can then be used to control the level of product-relatedspecies in the pooled material collected. In certain embodiments,specific signals arising from the chemical modification of the proteinssuch as glycation, MGO modification, deamidation, glycosylation may bespecifically measurable by spectroscopic methods through such in-line,on-line or at-line methods, enabling real time or near-real time controlof product quality of the resulting product.

In certain embodiments, the purification and/or pooling techniquesdescribed herein allow for the reduction of process-related impuritiesand/or other product-related substances. In certain embodiments, thepurification and/or pooling techniques described herein allow forreduction of process-related impurities and the selective inclusion ofparticular product-related substances. For example, but not by way oflimitation, the purification and/or pooling techniques described hereinallow for modulation of the concentration of product-related substancesin the purified sample, e.g., increasing or decreasing the amount of ARand/or basic species. In certain embodiments, the concentration ofparticular AR and/or basic species, e.g., Lys 0, Lys 1, and/or Lys 2,are modulated (increased or decreased) in the purified sample. Incertain embodiments, such techniques can be used to ensure productuniformity over the course of multiple production runs.

Anion Exchange (AEX) Chromatography

In certain embodiments, the low AR compositions of the invention areproduced by subjecting a primary protein recovery sample to at least oneanion exchange separation step after the above-described displacementchromatography step. In another embodiment, the anion exchange step willoccur before the above-described displacement chromatography step. Inone embodiment, the anion exchange chromatography step will occur afterthe above-described Protein A affinity and displacement chromatographysteps.

The use of an anionic exchange material versus a cationic exchangematerial, such as those cation exchange materials discussed in detailbelow, is based on the local charges of the protein of interest in agiven solution. Therefore, it is within the scope of this invention toemploy an anionic exchange step prior to the use of a cationic exchangestep, or a cationic exchange step prior to the use of an anionicexchange step. Furthermore, it is within the scope of this invention toemploy only an anionic exchange step, only an cationic exchange step, orany serial combination of the two (including serial combinations of oneor both ion exchange steps with the other chromatographic separationtechnologies described herein).

In performing the separation, the initial protein composition can becontacted with the anion exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique.

For example, in the context of batch purification, anion exchangematerial is prepared in, or equilibrated to, the desired startingbuffer. Upon preparation, or equilibration, a slurry of the anionexchange material is obtained. The protein of interest, e.g., antibody,solution is contacted with the slurry to allow for protein adsorption tothe anion exchange material. The solution comprising the acidic speciesthat do not bind to the AEX material is separated from the slurry, e.g.,by allowing the slurry to settle and removing the supernatant. Theslurry can be subjected to one or more washing steps and/or elutionsteps.

In the context of chromatographic separation, a chromatographicapparatus, commonly cylindrical in shape, is employed to contain thechromatographic support material (e.g., AEX material) prepared in anappropriate buffer solution. The chromatographic apparatus, ifcylindrical, can have a diameter of about 5 mm to about 2 meters, and aheight of 5 cm to 50 cm, and in certain embodiments, particularly forlarge scale processing, a height of ≦30 cm is employed. Once thechromatographic material is added to the chromatographic apparatus, asample containing the protein of interest, e.g., an antibody, iscontacted to the chromatographic material to induce the separation. Anyportion of the solution that does not bind to the chromatographicmaterial, e.g., which may comprise, depending on the AEX material beingemployed, the protein of interest, acidic species, is separated from thechromatographic material by washing the material and collectingfractions from column. The chromatographic material can be subjected toone or more wash steps. If desired, the chromatographic material canthen be contacted with a solution designed to desorb any components ofthe solution that have bound to the chromatographic material.

In certain embodiments, a wash step can be performed in the context ofAEX chromatography using conditions similar to the load conditions oralternatively by decreasing the pH and/or increasing the ionicstrength/conductivity of the wash in a step wise or linear gradientmanner. The resulting Flow Through and wash fractions can be analyzedand appropriate fractions pooled to achieve the desired reduction incharged variant species. In certain embodiments, the aqueous saltsolution used as both the loading and wash buffer has a pH that at ornear the isoelectric point (pI) of the protein of interest. In certainembodiments the pH is about 0 to 2 units higher or lower than the pI ofthe protein of interest. In certain embodiments, it will be in the rangeof 0 to 0.5 units higher or lower. In certain embodiments, it will be atthe pI of the antibody.

In certain non-limiting embodiments, the anionic agent is selected fromthe group consisting of acetate, formate, or combinations thereof. Incertain non-limiting embodiments, the cationic agent is selected fromthe group consisting of Tris, arginine, or combinations thereof. In oneembodiment, the buffer solution is a Tris/formate buffer. In anotherembodiment, the buffer is selected from the group consisting ofpyridine, piperazine, L-histidine, Bis-tris, Bis-tris propane,imidazole, N-Ethylmorpholine, TEA (triethanolamine), Tris, Morpholine,N-Methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol),diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propaol),piperazine, 1,3-Diaminopropane and piperidine.

A packed anion-exchange chromatography column, anion-exchange membranedevice, anion-exchange monolithic device, or depth filter media can beoperated either in bind-elute mode, flow-through mode, or a hybrid modewherein the product exhibits binding to the chromatographic material,yet can be washed from the column using a buffer that is the same orsubstantially similar to the loading buffer. In the bind-elute mode, thecolumn or the membrane device is first conditioned with a buffer withappropriate ionic strength and pH under conditions where certainproteins will be immobilized on the resin based matrix. For example, incertain embodiments, during the feed load, the protein of interest willbe adsorbed to the resin due to electrostatic attraction. After washingthe column or the membrane device with the equilibration buffer oranother buffer with different pH and/or conductivity, the productrecovery is achieved by increasing the ionic strength (i.e.,conductivity) of the elution buffer to compete with the solute for thecharged sites of the anion exchange matrix. Changing the pH and therebyaltering the charge of the solute is another way to achieve elution ofthe solute. The change in conductivity or pH may be gradual (gradientelution) or stepwise (step elution). In the flow-through mode, thecolumn or the membrane device is operated at selected pH andconductivity such that the protein of interest does not bind to theresin or the membrane while the acidic species will either be retainedon the column or will have a distinct elution profile as compared to theprotein of interest. In the context of this hybrid strategy, acidicspecies will bind to the chromatographic material (or Flow Through) in amanner distinct from the protein of interest, e.g., while the protein ofinterest and certain aggregates and/or fragments of the protein ofinterest may bind the chromatographic material, washes thatpreferentially remove the protein of interest can be applied. The columnis then regenerated before next use.

Non-limiting examples of anionic exchange substituents includediethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternaryamine (Q) groups. Additional non-limiting examples include: Poros 50PIand Poros 50HQ, which are a rigid polymeric bead with a backboneconsisting of cross-linked poly[styrene-divinylbenzene]; Capto Q Impresand Capto DEAE, which are a high flow agarose bead; Toyopearl QAE-550,Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are a polymericbase bead; Fractogel® EMD TMAE Hicap, which is a synthetic polymericresin with a tentacle ion exchanger; Sartobind STIC® PA nano, which is asalt-tolerant chromatographic membrane with a primary amine ligand;Sartobind Q nano; which is a strong anion exchange chromatographicmembrane; CUNO BioCap; which is a zeta-plus depth filter mediaconstructed from inorganic filter aids, refined cellulose, and an ionexchange resin; and X0HC, which is a depth-filter media constructed frominorganic filter aid, cellulose, and mixed cellulose esters. Thedetailed information is listed in Table A.

TABLE A List of AEX Adsorbent Properties Media Particle/ AEX AdsorbentVendor Type Ligand Type Pore Size Catalog Number Poros PI Applied ResinWeak ~50 μm 1-2459-11 Poros HQ Biosystems Strong ~50 μm 1-2559-11 CaptoDEAE GE Weak ~90 μm 17-5443-10 CaptoQ Impres Strong ~90 μm 17-5316-10QAE-550 Tosoh Strong ~100 μm 43271 DEAE-650 Weak ~65 μm 43201 GigaCapQ-650 Strong ~75 μm 21854 TMAE HiCap EMD/Millipore Strong ~40-90 μm1.16881.0013 Sartobind Sartorius Membrane Weak 3-5 μm 92STPA42DN- STIC ®PA Nano 11-A Sartobind Q Strong 3-5 μm 92IEXQ42DN-11 Nano CUNO BioCap 3MDepth NA NA BC0025L60ZA05A 25 Filter X0HC Millipore NA NA MX0HC23CL3

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 50 and 500 g/L, or between about 75 and 350 g/L, orbetween about 200 and 300 g/L. In certain embodiments, the proteinconcentration of the load protein mixture is adjusted to a proteinconcentration of the material loaded to the column of about 0.5 and 50g/L, between about 1 and 20 g/L, or between 3 and 10 g/L. In certainembodiments, the protein concentration of the load protein mixture isadjusted to a protein centration of the material to the column of about37 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR in the Flow Through and wash fractions while enriching for the samein the flow elution fraction, thereby producing protein compositionsthat have reduced AR or are free of AR. In certain embodiments relatingto the purification of adalimumab, the methods of the instant inventioncan be used to selectively remove, significantly reduce, or essentiallyremove all of AR1 charge variants in the Flow Through and wash fractionswhile enriching for the same in the flow elution fraction, therebyproducing protein compositions that have reduced AR1 or are free of AR1variants. In certain embodiments relating to adalimumab, the methods ofthe instant invention can be used to selectively remove, significantlyreduce, or essentially remove all of AR2 charge variants in theflow-through and wash fractions while enriching for the same in the flowelution fraction, thereby producing protein compositions that havereduced AR2 or are free of AR2 variants.

In certain embodiments, including but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe MGO variants in the Flow Through and wash fractions while enrichingfor the same in the elution fraction, thereby producing proteincompositions that have reduced MGO or are free of MGO variants (forexample, see U.S. Patent Application Ser. No. 61/777,883, filed on Mar.12, 2013). In certain embodiments, including, but not limited to thoserelating to adalimumab, the methods of the instant invention can be usedto selectively remove, significantly reduce, or essentially remove allof the glycated variants (Schiff's base and permanently glycated forms)in the Flow Through and wash fractions while enriching for the same inthe elution fraction, thereby producing protein preparations withreduced or free of glycated variants.

In certain embodiments, the loading, pH, conductivity of the AEXchromatography step, as well as elution pH conductivity, can be modifiedto achieve a desired distribution of product-relates substances (AR orlysine variants) For example, but not by way of limitation, certainembodiments are directed to the modulation of the lysine distribution ofpurified sample of a protein of interest, e.g., increasing Lys 0 anddecreasing Lys 1 and Lys 2. In certain embodiments, the methods of thepresent invention allow for the preparation of samples wherein theamount of Lys 0 is decreased, while the amount of Lys 1 and/or Lys 2 isincreased.

In certain embodiments, an AEX chromatographic separation can beperformed and combinations of fractions can be pooled to achieve acombination of desired process-related impurity and/or product-relatessubstance levels, in addition to, or in place of merely modulatingcharge variant concentration.

Spectroscopy methods such as UV, NIR, FTIR, Fluorescence, and Raman maybe used to monitor levels of AR species in an on-line, at-line orin-line mode, which can then be used to control the level of chargevariants, e.g., acidic species, in the pooled material collected fromthe AEX effluent.

In certain embodiments, specific signals arising from the chemicalmodification of the proteins such as glycation, MGO modification,deamidation, glycosylation may be specifically measurable byspectroscopic methods through such in-line, on-line or at-line methods,enabling realtime or near-real time control of product quality of theresulting product. In certain embodiments, on-line, at-line or in-linemonitoring methods can be used either on the effluent line of thechromatography step or in the collection vessel, to enable achievementof the desired product quality/recovery. In certain embodiments, the UVsignal can be used as a surrogate to achieve an appropriate productquality/recovery, wherein the UV signal can be processed appropriately,including, but not limited to, such processing techniques asintegration, differentiation, moving average, such that normal processvariability can be addressed and the target product quality can beachieved. In certain embodiments, such measurements can be combined within-line dilution methods such that ion concentration/conductivity of theload/wash can be controlled by feedback and hence facilitate productquality control.

In certain embodiments, a combination of AEX and CEX and MM methods canbe used to prepare product-related substance-modulated materials,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalintervening chromatography, filtration, pH adjustment, and/or UF/DFsteps so as to achieve the desired AR, product quality, ionconcentration, and/or viral reduction.

AEX chromatography can be used in conjunction with recyclechromatography modes and continuous chromatography modes.

Cation Exchange (CEX) Chromatography

The low AR compositions of the invention can be produced by subjectingthe composition, e.g., a primary recovery sample, to at least one cationexchange separation step after the above-described displacementchromatography step. In another embodiment, the cation exchange stepwill occur before the above-described displacement chromatography step.In one embodiment, the cation exchange chromatography step will occurafter the above-described Protein A affinity and displacementchromatography steps.

The use of a cationic exchange material versus an anionic exchangematerial, such as those anionic exchange materials discussed in detailabove, is based on the local charges of the protein of interest in agiven solution. Therefore, it is within the scope of this invention toemploy a cationic exchange step prior to the use of an anionic exchangestep, or an anionic exchange step prior to the use of a cationicexchange step. Furthermore, it is within the scope of this invention toemploy only a cationic exchange step, only an anionic exchange step, orany serial combination of the two (including serial combinations of oneor both ion exchange steps with the other chromatographic separationtechnologies described herein).

In performing the separation, the initial protein mixture can becontacted with the cation exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique, as described above in connection with ProteinA or AEX.

In certain embodiments, the aqueous salt solution used as both theloading and wash buffer has a pH that is lower than the isoelectricpoint (pI) of the protein of interest. In certain embodiments, the pH isabout 0 to 5 units lower than the pI of the protein. In certainembodiments, it is in the range of 1 to 2 units lower. In certainembodiments, it is in the range of 1 to 1.5 units lower.

In certain embodiments, the concentration of the anionic agent inaqueous salt solution is increased or decreased to achieve a pH ofbetween about 3.5 and 10.5, or between about 4 and 10, or between about4.5 and 9.5, or between about 5 and 9, or between about 5.5 and 8.5, orbetween about 6 and 8, or between about 6.5 and 7.5. In certainembodiments, the concentration of anionic agent is increased ordecreased in the aqueous salt solution to achieve a pH of 5, or 5.5, or6, or 6.5, or 6.8, or 7.5. Buffer systems suitable for use in the CEXmethods include, but are not limited to, tris formate, tris acetate,ammonium sulfate, sodium chloride and sodium sulfate.

In certain embodiments, the conductivity and pH of the aqueous saltsolution is adjusted by increasing or decreasing the concentration of acationic agent. In certain embodiments, the cationic agent is maintainedat a concentration of between about range of 20 mM to 500 mM, or betweenabout 50 to 350 mM or between about 100 to 300 mM or between about 100to 200 mM.

In certain non-limiting embodiments, the cationic agent is selected fromthe group consisting of sodium, Tris, tromethalmine, ammonium, arginine,or combinations thereof. In certain non-limiting embodiments, theanionic agent is selected from the group consisting of formate, acetate,citrate, chloride anion, sulphate, phosphate or combinations thereof.

A packed cation-exchange chromatography column or a cation-exchangemembrane device can be operated either in bind-elute mode, flow-throughmode, or a hybrid mode wherein the product exhibits binding to thechromatographic material, yet can be washed from the column using abuffer that is the same or substantially similar to the loading buffer.The details of these modes are outlined above.

Cationic substituents include carboxymethyl (CM), sulfoethyl (SE),sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional cationicmaterials include, but are not limited to: Capto SP ImpRes, which is ahigh flow agarose bead; CM Hyper D grade F; which is a ceramic beadcoated and permeated with a functionalized hydrogel, 250-400 ionicgroups μeq/mL; Eshmuno S, which is a hydrophilic polyvinyl ether basematrix with 50-100 μeq/mL ionic capacity; Nuvia C Prime, which is ahydrophobic cation exchange media composed of a macroporous highlycrosslinked hydrophilic polymer matrix 55-75 μeq/mL; Nuvia S, which hasa UNOsphere base matrix with 90-150 μeq/mL ionic groups; Poros HS; whichis a rigid polymetic bead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene]; Poros XS; which is a rigid polymetic beadwith a backbone consisting of cross-linked poly[styrene-divinylbenzene];Toyo Pearl Giga Cap CM 650M, which is a polymeric base bead with 0.225meq/mL ionic capacity; Toyo Pearl Giga Cap S 650M which is a polymericbase bead; Toyo Pearl MX TRP, which is a polymeric base bead. Detailedinformation concerning the aforementioned materials is listed in TableB. It is noted that CEX chromatography can be used with MM resins,described herein.

TABLE B Cationic Materials Catalog Resin Vendor Type Particle SizeNumber Capto SP ImpRes GE Strong ~40 μm 17-5468-10 CM Hyper D Pall Weak~50 μm 20050-027 Eshmuno S Millipore Strong ~85 μm 1.20078 Nuvia C PrimeBiorad Mix ~70 μm 156-3401 Mode Nuvia S Biorad Strong ~85 μm 156-0315Poros HS Applied Weak ~50 μm 13359-06 Biosystems Poros XS Applied Strong~50 μm 4404337 Biosystems Toyo Pearl Giga Cap CM Tosoh Weak ~75 μm 21946650M Toyo Pearl Giga Cap S Tosoh Strong ~75 μm 21833 650M Toyo Pearl MXTrp 650M Tosoh Mix ~75 μm 22817 Mode

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 5 and 150 g/L, or between about 10 and 100 g/L, betweenabout 20 and 80 g/L, between about 30 and 50 g/L, or between about 40and 50 g/L. In certain embodiments, the protein concentration of theload protein mixture is adjusted to a protein concentration of thematerial loaded to the column of about 0.5 and 50 g/L, or between about1 and 20 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR in the Flow Through and wash fractions while enriching for the samein the elution fraction, thereby producing protein compositions thathave reduced AR or are free of AR. In certain embodiments relating tothe purification of adalimumab, the methods of the instant invention canbe used to selectively remove, significantly reduce, or essentiallyremove all of AR1 charge variants in the Flow Through and wash fractionswhile enriching for the same in the flow elution fraction, therebyproducing protein compositions that have reduced AR1 or are free of AR1variants. In certain embodiments relating to adalimumab, the methods ofthe instant invention can be used to selectively remove, significantlyreduce, or essentially remove all of AR2 charge variants in theflow-through and wash fractions while enriching for the same in the flowelution fraction, thereby producing protein compositions that havereduced AR2 or are free of AR2 variants.

In certain embodiments, including, but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe MGO variants in the elution fractions while enriching for the samein the Flow Through and wash fractions, thereby producing proteinpreparations with reduced or free of MGO variants. In certainembodiments, including, but not limited to those relating to adalimumab,the methods of the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of the glycated variants(Schiff's base and permanently glycated forms) in the elution fractionswhile enriching for the same in the Flow Through and wash fractions,thereby producing protein preparations with reduced or free of glycatedvariants.

In certain embodiments, the loading, pH, conductivity of the CEXchromatography step, as well as elution pH conductivity, can be modifiedto achieve a desired distribution of acidic species. For example, butnot by way of limitation, certain embodiments are directed to themodulation of the lysine distribution of a purified sample of a proteinof interest, e.g., increasing Lys 0 and decreasing Lys 1 and Lys 2. Incertain embodiments, the methods of the present invention allow for thepreparation of samples wherein the amount of Lys 0 is decreased, whilethe amount of Lys 1 and/or Lys 2 is increased.

In certain embodiments, a CEX chromatographic separation can beperformed and combinations of fractions can be pooled to achieve acombination of desired process-related impurity and/or product-relatessubstance levels, in addition to, or in place of merely modulatingcharge variant concentration.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of product-relatedcharge variants, aggregates, low molecular weight variants (e.g.,fragments of the protein of interest) in an on-line, at-line or in-linemode, which can then be used to control the level of charge variants,e.g., acidic species, in the pooled material collected from the CEXeffluent. In certain embodiments, specific signals arising from thechemical modification of the proteins such as glycation, MGOmodification, deamidation, glycosylation may be specifically measurableby spectroscopic methods through such in-line, on-line or at-linemethods, enabling realtime or near-real time control of product qualityof the resulting product. In certain embodiments, on-line, at-line orin-line monitoring methods can be used either on the effluent line ofthe chromatography step or in the collection vessel, to enableachievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, moving average, such thatnormal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control.

In certain embodiments, a combination of CEX and AEX and/or MM methodscan be used to prepare product-related substance-modulated materials,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalchromatography, filtration, pH adjustment, UF/DF steps so as to achievethe desired product quality, AR, ion concentration, and/or viralreduction.

CEX chromatography can be used in conjunction with recyclechromatography and continuous chromatography modes.

Mixed Mode Chromatography

Mixed mode (“MM”) chromatography may also be used to prepare the low ARcompositions of the invention. MM chromatography, also referred toherein as “multimodal chromatography”, is a chromatographic strategythat utilizes a support comprising a ligand that is capable of providingat least two different, and in certain embodiments co-operative, sitesthat interact with the substance to be bound. In certain embodiments,one of these sites gives an attractive type of charge-charge interactionbetween the ligand and the substance of interest and the other siteprovides for electron acceptor-donor interaction and/or hydrophobicand/or hydrophilic interactions. Electron donor-acceptor interactionsinclude interactions such as hydrogen-bonding, π-π, cation-π, chargetransfer, dipole-dipole, induced dipole etc.

In certain embodiments, the resin employed for a mixed mode separationis Capto Adhere. Capto Adhere is a strong anion exchanger withmultimodal functionality. Its base matrix is a highly cross-linkedagarose with a ligand (N-Benzyl-N-methyl ethanol amine) that exhibitsmany functionalities for interaction, such as ionic interaction,hydrogen bonding and hydrophobic interaction. In certain embodiments,the resin employed for a mixed mode separation is selected fromPPA-HyperCel and HEA-HyperCel. The base matrices of PPA-HyperCel andHEA-HyperCel are high porosity cross-linked cellulose. Their ligands arePhenylpropylamine and Hexylamine, respectively. Phenylpropylamine andHexylamine offer different selectivity and hydrophobicity options forprotein separations. Additional mixed mode chromatographic supportsinclude, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M,and Eshmuno® HCX.

In certain embodiments, the mixed mode chromatography resin is comprisedof ligands coupled to an organic or inorganic support, sometimes denoteda base matrix, directly or via a spacer. The support may be in the formof particles, such as essentially spherical particles, a monolith,filter, membrane, surface, capillaries, etc. In certain embodiments, thesupport is prepared from a native polymer, such as cross-linkedcarbohydrate material, such as agarose, agar, cellulose, dextran,chitosan, konjac, carrageenan, gellan, alginate etc. To obtain highadsorption capacities, the support can be porous, and ligands are thencoupled to the external surfaces as well as to the pore surfaces. Suchnative polymer supports can be prepared according to standard methods,such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta79(2), 393-398 (1964). Alternatively, the support can be prepared from asynthetic polymer, such as cross-linked synthetic polymers, e.g. styreneor styrene derivatives, divinylbenzene, acrylamides, acrylate esters,methacrylate esters, vinyl esters, vinyl amides etc. Such syntheticpolymers can be produced according to standard methods, see e.g.“Styrene based polymer supports developed by suspension polymerization”(R Arshady: Chimica e L′Industria 70(9), 70-75 (1988)). Porous native orsynthetic polymer supports are also available from commercial sources,such as Amersham Biosciences, Uppsala, Sweden.

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 50 and 750 g/L, or between about 75 and 500 g/L, orbetween about 100 and 300 g/L. In certain embodiments, the proteinconcentration of the load protein mixture is adjusted to a proteinconcentration of the material loaded to the column of about 1 and 50g/L, or between about 9 and 25 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toadalimumab, the MM methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR in the Flow Through and wash fractions while enriching for the samein the flow elution fraction, thereby producing protein compositionsthat have reduced AR or are free of AR. In certain embodiments relatingto the purification of adalimumab, the methods of the instant inventioncan be used to selectively remove, significantly reduce, or essentiallyremove all of AR1 charge variants in the Flow Through and wash fractionswhile enriching for the same in the flow elution fraction, therebyproducing protein compositions that have reduced AR1 or are free of AR1variants. In certain embodiments relating to adalimumab, the methods ofthe instant invention can be used to selectively remove, significantlyreduce, or essentially remove all of AR2 charge variants in theflow-through and wash fractions while enriching for the same in the flowelution fraction, thereby producing protein compositions that havereduced AR2 or are free of AR2 variants.

In certain embodiments, including, but not limited to those relating toadalimumab, the MM methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe MGO variants in the Flow Through and wash fractions while enrichingfor the same in the elution fraction, thereby producing proteinpreparations with reduced or free of MGO variants. In certainembodiments, including, but not limited to those relating to adalimumab,the methods of the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of the glycated variants(Schiff's base and permanently glycated forms) in the Flow Through andwash fractions while enriching for the same in the elution fraction,thereby producing protein preparations with reduced or free of glycatedvariants.

In certain embodiments, the loading, pH, conductivity of the MMchromatography step, wash pH and conductivity, as well as elution pHconductivity, can be modified to achieve a desired distribution ofacidic species. For example, but not by way of limitation, certainembodiments are directed to the modulation of the lysine distribution ofa purified sample of a protein of interest, e.g., increasing Lys 0 anddecreasing Lys 1 and Lys 2. In certain embodiments, the methods of thepresent invention allow for the preparation of samples wherein theamount of Lys 0 is decreased, while the amount of Lys 1 and/or Lys 2 isincreased.

In certain embodiments, a MM chromatographic separation can be performedand combinations of fractions can be pooled to achieve a combination ofdesired process-related impurity and/or product-relates substancelevels, in addition to, or in place of merely modulating charge variantconcentration.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of AR species in anon-line, at-line or in-line mode, which can then be used to control thelevel of charge variants, e.g., acidic species, in the pooled materialcollected from the MM effluent. In certain embodiments, specific signalsarising from the chemical modification of the proteins such asglycation, MGO modification, deamidation, glycosylation may bespecifically measurable by spectroscopic methods through such in-line,on-line or at-line methods, enabling real time or near-real time controlof product quality of the resulting product. In certain embodiments,on-line, at-line or in-line monitoring methods can be used either on theeffluent line of the chromatography step or in the collection vessel, toenable achievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, moving average, such thatnormal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control.

In certain embodiments, a combination of mixed mode and AEX and CEXmethods can be used to prepare the low AR compositions of the invention,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalintervening chromatography, filtration, pH adjustment, UF/DF steps so asto achieve the desired product quality, AR, ion concentration, and/orviral reduction.

MM chromatography can be used in conjunction with recycle chromatographyand continuous chromatography modes.

Continuous and Recycle Chromatography

Continuous and recycle chromatography modes can be used to produce thelow AR compositions of the invention, and are described below. Thesemethods result in significant improvements in recovery of the protein,e.g., antibody, of interest while maintaining the AR reduction levels.These continuous and recycle chromatography modes are applicable tochromatography methods where (a) the low acidic species component ofinterest is collected in the unbound fraction during the chromatography(Flow Through/wash chromatography) or (b) where the low acidic speciescomponent of interest is first bound to the media and subsequentlyrecovered by washing the media with conditions that elute the boundcomponent.

Continuous and Recycle Chromatography—Flow Through/Wash Chromatography

In the case where the low acidic species component of interest iscollected in the unbound fraction, the following approach is employedwhich prevents loss of the material loaded on the column.

In one embodiment, a recycle chromatography mode is used wherein thecolumn is loaded and the unbound fractions that results in the target ARlevel are collected. Subsequently, instead of regenerating the columnand losing the product, the column is washed under conditions thatresult in recovery of the product remaining bound to the column. Thisproduct recovered under these conditions contains significantly higherAR levels than the original feed material. This wash fraction isadjusted to the appropriate conditions to achieve the separation desiredon subsequent processing (typically similar conditions to the initialpreparation) and combined with the original feed material and loaded onthe column again (after preparing the column appropriately for the nextcycle). The amount of material prepared for the next cycle, combiningthe wash fraction from the first cycle and the fresh material isadjusted to the target loading capacity for the column to achieve thedesired separation (typically similar to the capacity targeted for thefirst cycle).

In performing the second cycle, a similar strategy is employed,collecting the unbound fraction so as to achieve the target AR level andthen subsequently washing the column under conditions to recover theproduct remaining on the column.

In one embodiment, this recycle chromatography mode is continued untilall the load materials are used. The number of cycles can be controlledby designing the column size appropriately.

In employing the recycle chromatography mode, the recovery of theproduct loaded on the column is significantly improved while achievingthe target AR levels.

Several variations of the recycle chromatography mode can be employed.In one embodiment, the fractions that are collected targeting a certainAR level can be determined based on predetermined criteria or based onat-line, off-line or on-line analysis of the effluent of the column orthe collected pool.

In another embodiment, the wash conditions used for the first cycle canbe adjusted to recover the desired amount of product at the desiredproduct quality, only limited by the feasibility of preparing anappropriate load mixture for the subsequent step. In one aspect of thisembodiment, the wash condition may be similar to the load condition. Inanother aspect of this embodiment, the wash condition can be stringentto recover all of the product species (desired and undesired) remainingon the column.

In still another embodiment, the loading amount, the loading conditionsand the washing conditions used for the subsequent cycles can bemodified to achieve the desired purity, given that that loading materialfor the subsequent cycles are likely to contain higher levels of AR.

In another embodiment, the last cycle of the operation can be performedunder different conditions such that the target purity and targetrecovery can be achieved to optimize overall recovery and purity.

The methods for producing the low AR composition of the invention canalso be implemented in a continuous chromatography mode. In this mode,at least two columns are employed (referred to as a “first” column and a“second” column). In one embodiment, the feed material is loaded ontothe first column, and the unbound fraction from the first column iscollected such that the pool material contains the target AR level. Thecolumn is then washed under conditions that recover the remainingproduct. This material is then dynamically diluted with appropriatesolutions to achieve the desired loading conditions, mixed with freshfeed material and directed to the second column. The unbound fractionfrom the second column is collected to achieve the target AR level. Thesecond column is then washed under conditions to recover the product anddiluted with appropriate solutions, mixed with fresh materialsdynamically and directed to the first column (which is prepared toreceive the load after regeneration/cleaning). In one embodiment, thiscycling is continued until all the load material is used. The last cyclecan be operated in a “typical” mode, with appropriate adjustments to theload and wash conditions as necessary.

In certain embodiments this continuous chromatography mode can becarried out such that the wash material containing the higher AR levelscan be directed back into the load tank after appropriate dilution. Thismaterial can then be loaded subsequently or concurrently onto the secondcolumn, such that the operation of the two columns are not in tandem,reducing complexity of the operation.

This continuous chromatography mode, while similar to the recyclechromatography mode, can be carried out more efficiently, and thereforehas a reduced processing time.

For this continuous chromatography mode, several variations can beemployed. In one embodiment, the fractions that are collected targetinga certain AR level can be determined based on predetermined criteria orbased on at-line, off-line or on-line analysis of the effluent of thecolumn or the collected pool.

In another embodiment, the wash conditions used for the first cycle canbe adjusted to recover the desired amount of product at the desiredproduct quality, only limited by the feasibility of preparing anappropriate load mixture for the subsequent step. In one aspect of thisembodiment, the wash conditions may be similar to the load conditions.In another aspect of the embodiment, the wash conditions can bestringent to recover all of the product species (desired and undesired)remaining on the column.

In still another embodiment, the loading amount, the loading conditionsand the washing conditions used for the subsequent cycles can bemodified to achieve the desired purity, given that that loading materialfor the subsequent cycles are likely to contain higher levels of AR.

In another embodiment, the last cycle of the operation can be performedunder different conditions such that the target purity/recovery can beachieved to optimize overall recovery and and/or purity.

In one embodiment, the media choice for the recycle or continuous modescan be one of many chromatographic resins with pendant hydrophobic andanion exchange functional groups, monolithic media, membrane adsorbentmedia or depth filtration media.

In certain embodiments, membrane or depth filter based media(“convective media”) can be used in the recycle or continuouschromatography modes because selectivity of separation is not requiredto be high given the fact that the less enriched portions of the productare “recycled” while the pure fractions are selectively pooled.

Continuous and Recycle Chromatography—Elution Chromatography

In the elution mode of chromatography or separation, as exemplified bythe CEX technology for AR reduction, the conditions are chosen for theload and wash steps such that the AR enriched material is collected inthe Flow Through and/or wash fractions, while the AR reduced material iscollected in the elution fraction. In the typical implementation of theCEX technology, about 10 to 40% of the product (the desired chargevariant) may be lost in the Flow Through/Wash fractions. Two modes ofoperation, namely the recycle chromatography mode and the continuouschromatography mode provide improved recovery, while maintaining thetarget AR levels.

In the recycle chromatography mode, the load material is, in general,processed over multiple cycles. In implementing the recyclechromatography mode, the load material is prepared such that the eluatecontains the target product purity or AR level. Under these conditions,the AR enriched material is collected in the Flow Through/washfractions. This material is pooled and additional fresh load material isadded to achieve the appropriate loading capacity for the next cycle ofchromatography on the same column. In particular, in one embodiment, thecolumn is eluted under conditions where the bound product (having low ARlevels) is recovered, and subsequently regenerated and equilibrated toprepare for the next cycle.

In the next cycle, the combined load (Flow Through/wash from cycle 1above, as well as fresh material) is loaded to the target capacity. TheFlow Through/wash fractions are collected and pooled. The column iseluted to obtain the second eluate, again containing the target low ARcomposition. In one embodiment, this sequence is continued until all theload materials are processed.

In another embodiment, by implementing the recycle chromatography mode,the material that would otherwise be discarded as AR enriched materialis further purified to “recover” pure protein product, thereby improvingthe overall recovery of the protein. In one embodiment, the level ofrecovery depends on the number of cycles employed.

For the recycle chromatography mode, several variations can be employed.In one embodiment, the entire pool of the Flow Through/wash fractionsare preferably combined with fresh materials to maximize recovery of theentire operation. However, a portion of the flow through wash can bediscarded to achieve higher purity or efficiency. For example, in oneembodiment, certain fractions containing very high levels of AR speciescan be discarded. To enable such selective pooling, off-line, in-line orat line methods can be used to directly or indirectly measure the levelsof AR.

In another embodiment, the loading amount and the conditions forloading, washing and eluting can be modified for the second andsubsequent cycles to accommodate the higher levels of AR that will bepresent in the loading pool.

In still another embodiment, the last cycle of the method can beperformed under conditions such that the target purity and recovery canbe achieved to optimize overall recovery and purity.

A continuous chromatography mode provides additional advantages in termsof time efficiency. In this mode of operation, two or more columns areused. Specifically, as with the recycle mode, an appropriate conditionfor the load capacity, load, wash and elution conditions are chosen forthe operation. The Flow Through and wash fractions (or a portionthereof) is directed to the load tank containing the fresh material.After completion of the load and wash steps, the first column is elutedand subsequently regenerated. Meanwhile, the second column is loadedwith the material that is a mix of fresh material and the wash and FlowThrough from the previous cycle. The wash and Flow Through from thesecond column is again directed back to the load tank. The second columnis then eluted and regenerated. The first column is then ready to beloaded and the cycle continues. This continuous chromatography mode isefficient as the product is processed continuously and the purifiedproduct is obtained in a semi-continuous manner.

Several variations of the continuous chromatography mode can beemployed. In one embodiment, the entire pool of the Flow Through/washfractions is combined with fresh materials to maximize recovery of theentire operation. However, a portion of the Flow Through wash can bediscarded to achieve higher purity or efficiency. For example, certainfractions containing very high levels of AR species can be discarded. Toenable such selective pooling, off-line, in-line or at line methods canbe used to measure directly or indirectly the levels of acidic species.

In another embodiment, the loading amount, conditions for loading,washing and eluting can be modified for the second and subsequent cyclesto accommodate the higher levels of AR that will be present in theloading pool.

In still another embodiment, the last cycle of the operation can beperformed under different conditions to optimize overall recover andpurity.

The recycle chromatography mode and the continuous chromatography modeare not limited to use with any particular chromatography resin. Themedia used for the recycle or continuous modes can be one of manychromatographic resins with pendant hydrophobic and anion exchangefunctional groups, monolithic media, membrane adsorber media or depthfiltration media.

In certain embodiments, membrane depth filter-based media (“convectivemedia”) can be used with the recycle or continuous modes as theselectivity of separation is not required to be high given the fact thatthe less enriched portions of the product are “recycled” while the purefractions are selectively pooled.

Recycle chromatography mode and the continuous chromatography mode canbe used inconjunction with AEX, CEX, or MM chromatography methods, asdescribed herein, to produce the low AR compositions of the invention.

Hydrophobic Interaction Chromatography

The low AR compositions of the invention may also be prepared using ahydrophobic interaction chromatography (HIC) step in addition to thedisplacement chromatography step.

In performing the separation, the sample mixture is contacted with theHIC material, e.g., using a batch purification technique or using acolumn or membrane chromatography. Prior to HIC purification it may bedesirable to adjust the concentration of the salt buffer to achievedesired protein binding to the resin or the membrane.

Whereas ion exchange chromatography relies on the local charge of theprotein of interest for selective separation, hydrophobic interactionchromatography employs the hydrophobic properties of the proteins toachieve selective separation. Hydrophobic groups on the protein interactwith hydrophobic groups of the resin or the membrane. The morehydrophobic a protein is the stronger it will interact with the columnor the membrane. Thus the HIC step removes process-related impurities(e.g., HCPs) as well as product-related substances (e.g., aggregates andfragments).

Like ion exchange chromatography, a HIC column or membrane device canalso be operated in product a bind-elute mode, a flow-through, or ahybrid mode wherein the product exhibits binding to the chromatographicmaterial, yet can be washed from the column using a buffer that is thesame or substantially similar to the loading buffer. The details ofthese modes are outlined above in connection with AEX purification.

As hydrophobic interactions are strongest at high ionic strength, thisform of separation is conveniently performed following salt elutionstep, such as those that are typically used in connection with ionexchange chromatography. Alternatively, salts can be added into a lowsalt level feed stream before this step. Adsorption of the antibody to aHIC column is favored by high salt concentrations, but the actualconcentrations can vary over a wide range depending on the nature of theprotein of interest, salt type and the particular HIC ligand chosen.Various ions can be arranged in a so-called soluphobic series dependingon whether they promote hydrophobic interactions (salting-out effects)or disrupt the structure of water (chaotropic effect) and lead to theweakening of the hydrophobic interaction. Cations are ranked in terms ofincreasing salting out effect as Ba²⁺; Ca²⁺; Me⁺; Li⁺; Cs⁺; Na⁺; K⁺;Rb⁺; NH₄ ⁺, while anions may be ranked in terms of increasing chaotropiceffect as PO₄ ³⁻; SO₄ ²⁻; CH₃CO₃ ⁻; Cr; Br⁻; NO₃ ⁻; ClO₄ ⁻; I⁻; SCN⁻.

In general, Na⁺, K⁺ or NH₄ ⁺ sulfates effectively promote ligand-proteininteraction in HIC. Salts may be formulated that influence the strengthof the interaction as given by the following relationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr>NaSCN. In general, salt concentrationsof between about 0.75 M and about 2 M ammonium sulfate or between about1 and 4 M NaCl are useful.

HIC media normally comprise a base matrix (e.g., cross-linked agarose orsynthetic copolymer material) to which hydrophobic ligands (e.g., alkylor aryl groups) are coupled. A suitable HIC media comprises an agaroseresin or a membrane functionalized with phenyl groups (e.g., a PhenylSepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). ManyHIC resins are available commercially. Examples include, but are notlimited to, Capto Phenyl, Phenyl Sepharose™ 6 Fast Flow with low or highsubstitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ HighPerformance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMDPhenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butylcolumns (Bio-Rad, California); WP HI-Propyl (C3)™ (J. T. Baker, NewJersey); and Toyopearl™ ether, phenyl or butyl (TosoHaas, PA).

Viral Filtration

Viral filtration is a dedicated viral reduction step in the entirepurification process. This step is usually performed postchromatographic polishing steps. Viral reduction can be achieved via theuse of suitable filters including, but not limited to, Planova 20N™, 50N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMDMillipore, ViroS art CPV from Sartorius, or Ultipor DV20 or DV50™ filterfrom Pall Corporation. It will be apparent to one of ordinary skill inthe art to select a suitable filter to obtain desired filtrationperformance.

Ultrafiltration/Diafiltration

Certain embodiments of the present invention employ ultrafiltration anddiafiltration steps to further concentrate and formulate the protein ofinterest, e.g., an antibody product, in addition to the displacementchromatography steps. Ultrafiltration is described in detail in:Microfiltration and Ultrafiltration: Principles and Applications, L.Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and in:Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986;ISBN No. 87762-456-9). One filtration process is Tangential FlowFiltration as described in the Millipore catalogue entitled“Pharmaceutical Process Filtration Catalogue” pp. 177-202 (Bedford,Mass., 1995/96). Ultrafiltration is generally considered to meanfiltration using filters with a pore size of smaller than 0.1 μm. Byemploying filters having such small pore size, the volume of the samplecan be reduced through permeation of the sample buffer through thefilter membrane pores while proteins, such as antibodies, are retainedabove the membrane surface.

Diafiltration is a method of using membrane filters to remove andexchange salts, sugars, and non-aqueous solvents, to separate free frombound species, to remove low molecular-weight species, and/or to causethe rapid change of ionic and/or pH environments. Microsolutes areremoved most efficiently by adding solvent to the solution beingdiafiltered at a rate approximately equal to the permeate flow rate.This washes away microspecies from the solution at a constant volume,effectively purifying the retained protein of interest. In certainembodiments of the present invention, a diafiltration step is employedto exchange the various buffers used in connection with the instantinvention, optionally prior to further chromatography or otherpurification steps, as well as to remove impurities from the proteinpreparations.

One of ordinary skill in the art can select appropriate membrane filterdevice for the UF/DF operation. Examples of membrane cassettes suitablefor the present invention include, but not limited to, Pellicon 2 orPellicon 3 cassettes with 10 kD, 30kD or 50 kD membranes from EMDMillipore, Kvick 10 kD, 30 kD or 50 kD membrane cassettes from GEHealthcare, and Centramate or Centrasette 10 kD, 30 kD or 50 kDcassettes from Pall Corporation.

Exemplary Purification Strategies

In certain embodiments, primary recovery can proceed by sequentiallyemploying pH reduction, centrifugation, and filtration steps to removecells and cell debris (including HCPs) from the production bioreactorharvest. In certain embodiments, the present invention is directed tosubjecting a sample mixture from said primary recovery to Protein Aaffinity followed by displacement chromatography. Certain embodiments ofthe present invention will include further purification steps. Examplesof additional purification procedures which can be performed prior to,during, or following the displacement chromatography method includeethanol precipitation, isoelectric focusing, reverse phase HPLC,chromatography on silica, chromatography on heparin Sepharose™, furtheranion exchange chromatography and/or further cation exchangechromatography, chromatofocusing, SDS-PAGE, ammonium sulfateprecipitation, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography (e.g., using protein G, anantibody, a specific substrate, ligand or antigen as the capturereagent).

Specific examples of such combinations of strategies is presented below,with specific data relating to particular combinations useful in thecontext of the instant invention included in Examples 1-8 and in Tables80-87 and 76-78 of U.S. Provisional Application No. 61/893,068, entitled“Low Acidic Species Compositions and Methods for Producing and Using theSame”, filed on Oct. 18, 2013, the entire contents of which areexpressly incorporated herein by reference.

In certain embodiments, the unbound Flow Through and wash fractions canbe further fractionated and a combination of fractions providing atarget product purity can be pooled.

In certain embodiments, the protein concentration can be adjusted toachieve a differential partitioning behavior between the antibodyproduct and the product-related substances, such that the purity and/oryield can be further improved. In certain embodiments, the loading canbe performed at different protein concentrations during the loadingoperation to improve the product quality/yield of any particularpurification step.

In certain embodiments, the column temperature can be independentlyvaried to improve the separation efficiency and/or yield of anyparticular purification step.

In certain embodiments, the loading and washing buffer matrices can bedifferent or composed of mixtures of chemicals, while achieving similar“resin interaction” behavior such that the above novel separation can beeffected. For example, but not by way of limitation, the loading andwashing buffers can be different, in terms of ionic strength or pH,while remaining substantially similar in function in terms of thewashout of the product achieved during the wash step. In certainembodiments, additives such as amino acids, sugars, PEG, etc can beadded to the load or wash steps to modulate the partitioning behavior toachieve the separation efficiency and/or yield.

In certain embodiments, the loading & washing steps can be controlled byin-line, at-line or off-line measurement of the product relatedimpurity/substance levels, either in the column effluent, or thecollected pool or both, so as to achieve the target product qualityand/or yield. In certain embodiments, the loading concentration can bedynamically controlled by in-line or batch or continuous dilutions withbuffers or other solutions to achieve the partitioning necessary toimprove the separation efficiency and/or yield.

IV. METHODS OF ASSAYING SAMPLE PURITY

Assaying Host Cell Protein

The present invention also provides methods for determining the residuallevels of host cell protein (HCP) concentration in the low ARcompositions of the invention. As described above, HCPs are desirablyexcluded from the final target substance product. Exemplary HCPs includeproteins originating from the source of the antibody production. Failureto identify and sufficiently remove HCPs from the target antibody maylead to reduced efficacy and/or adverse reactions in a subject.

As used herein, the term “HCP ELISA” refers to an ELISA where the secondantibody used in the assay is specific to the HCPs produced from cells,e.g., CHO cells, used to generate the antibody of interest. The secondantibody may be produced according to conventional methods known tothose of skill in the art. For example, the second antibody may beproduced using HCPs obtained by sham production and purification runs,i.e., the same cell line used to produce the antibody of interest isused, but the cell line is not transfected with antibody DNA. In anexemplary embodiment, the second antibody is produced using HCPs similarto those expressed in the cell expression system of choice, i.e., thecell expression system used to produce the target antibody.

Generally, HCP ELISA comprises sandwiching a liquid sample comprisingHCPs between two layers of antibodies, i.e., a first antibody and asecond antibody. The sample is incubated during which time the HCPs inthe sample are captured by the first antibody, for example, but notlimited to goat anti-CHO, affinity purified (Cygnus). A labeled secondantibody, or blend of antibodies, specific to the HCPs produced from thecells used to generate the antibody, e.g., anti-CHO HCP Biotinylated, isadded, and binds to the HCPs within the sample. In certain embodimentsthe first and second antibodies are polyclonal antibodies. In certainaspects the first and second antibodies are blends of polyclonalantibodies raised against HCPs. The amount of HCP contained in thesample is determined using the appropriate test based on the label ofthe second antibody.

HCP ELISA may be used for determining the level of HCPs in an antibodycomposition, such as an eluate or flow-through obtained using theprocess described above. The present invention also provides acomposition comprising an antibody, wherein the composition has nodetectable level of HCPs as determined by an HCP Enzyme LinkedImmunosorbent Assay (“ELISA”).

Assaying Acidic Species (AR)

The levels of acidic species in the chromatographic samples producedusing the techniques described herein may be analyzed as described inthe Examples section. In certain embodiments a CEX-HPLC method isemployed. For example, but not by way of limitation, cation exchangechromatography can be performed on a Dionex ProPac WCX-10, Analyticalcolumn 4 mm×250 mm (Dionex, CA). An Agilent 1200 HPLC system can then beused as the HPLC. In certain embodiments, mobile phases such as 10 mMSodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM SodiumPhosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B) can beused. In certain embodiments, a binary gradient (94% A, 6% B: 0-20 min;84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34min) can be used with detection at 280 nm. In certain embodiments,quantitation is based on the relative area percent of detected peaks. Incertain embodiments, the peaks that elute at relative residence timeless than a certain time are together represented as the acidic peaks.

Assaying Size Variants

In certain embodiments, the levels of aggregates, monomer, and fragmentsin the chromatographic samples produced using the techniques describedherein are analyzed. In certain embodiments, the aggregates, monomer,and fragments are measured using a size exclusion chromatographic (SEC)method for each molecule. For example, but not by way of limitation, aTSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300 mm column (Tosoh Bioscience) canbe used in connection with certain embodiments, while a TSK-gel SuperSW3000, 4 μm, 250 Å, 4.6×300 mm column (Tosoh Bioscience) can be used inalternative embodiments. In certain embodiments, the aforementionedcolumns are used along with an Agilent or a Shimazhu HPLC system. Incertain embodiments, sample injections are made under isocratic elutionconditions using a mobile phase consisting of, for example, 100 mMsodium sulfate and 100 mM sodium phosphate at pH 6.8, and detected withUV absorbance at 214 nm. In certain embodiments, the mobile phase willconsist of 1×PBS at pH 7.4, and elution profile detected with UVabsorbance at 280 nm. In certain embodiments, quantification is based onthe relative area of detected peaks.

Any additional technique, such as mass spectroscopy, can be used forassaying size variants.

V. METHODS OF TREATMENT USING THE LOW AR COMPOSITIONS OF THE INVENTION

The low AR compositions of the invention may be used to treat anydisorder in a subject for which the therapeutic protein comprised in thecomposition is appropriate for treating. \

A “disorder” is any condition that would benefit from treatment with theprotein. This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the subject to thedisorder in question. In the case of an anti-TNFα antibody, or antigenbinding portion thereof, such as adalimumab, a therapeutically effectiveamount of the low AR composition may be administered to treat a disorderin which TNFα activity is detrimental.

A disorder in which TNFα activity is detrimental includes a disorder inwhich inhibition of TNFα activity is expected to alleviate the symptomsand/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNFα in a biologicalfluid of a subject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject), which can be detected, for example, using an anti-TNFαantibody.

TNFα has been implicated in the pathophysiology of a wide variety of aTNFα-related disorders including sepsis, infections, autoimmunediseases, transplant rejection and graft-versus-host disease (see e.g.,Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024to Moeller et al.; European Patent Publication No. 260 610 B1 byMoeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452;Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).Accordingly, the low AR compositions or a low process-related impuritycompositions of the invention may be used to treat an autoimmunedisease, such as rheumatoid arthritis, juvenile idiopathic arthritis, orpsoriatic arthritis, an intestinal disorder, such as Crohn's disease orulcerative colitis, a spondyloarthropathy, such as ankylosingspondylitis, or a skin disorder, such as psoriasis.

Disorders in which TNFα activity is detrimental are well known in theart and described in detail in U.S. Pat. No. 8,231,876 and U.S. Pat. No.6,090,382, the entire contents of each of which are expresslyincorporated herein by reference. In one embodiment, “a disorder inwhich TNFα activity is detrimental” includes sepsis (including septicshock, endotoxic shock, gram negative sepsis and toxic shock syndrome),autoimmune diseases (including rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis, nephrotic syndrome,multisystem autoimmune diseases, lupus (including systemic lupus, lupusnephritis and lupus cerebritis), Crohn's disease and autoimmune hearingloss), infectious diseases (including malaria, meningitis, acquiredimmune deficiency syndrome (AIDS), influenza and cachexia secondary toinfection), allograft rejection and graft versus host disease,malignancy, pulmonary disorders (including adult respiratory distresssyndrome (ARDS), shock lung, chronic pulmonary inflammatory disease,pulmonary sarcoidosis, pulmonary fibrosis, silicosis, idiopathicinterstitial lung disease and chronic obstructive airway disorders(COPD), such as asthma), intestinal disorders (including inflammatorybowel disorders, idiopathic inflammatory bowel disease, Crohn's diseaseand Crohn's disease-related disorders (including fistulas in thebladder, vagina, and skin; bowel obstructions; abscesses; nutritionaldeficiencies; complications from corticosteroid use; inflammation of thejoints; erythem nodosum; pyoderma gangrenosum; lesions of the eye,Crohn's related arthralgias, fistulizing Crohn's indeterminant colitisand pouchitis), cardiac disorders (including ischemia of the heart,heart insufficiency, restenosis, congestive heart failure, coronaryartery disease, angina pectoris, myocardial infarction, cardiovasculartissue damage caused by cardiac arrest, cardiovascular tissue damagecaused by cardiac bypass, cardiogenic shock, and hypertension,atherosclerosis, cardiomyopathy, coronary artery spasm, coronary arterydisease, valvular disease, arrhythmias, and cardiomyopathies),spondyloarthropathies (including ankylosing spondylitis, psoriaticarthritis/spondylitis, enteropathic arthritis, reactive arthritis orReiter's syndrome, and undifferentiated spondyloarthropathies),metabolic disorders (including obesity and diabetes, including type 1diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy,peripheral neuropathy, diabetic retinopathy, diabetic ulcerations,retinopathy ulcerations and diabetic macrovasculopathy), anemia, pain(including acute and chronic pains, such as neuropathic pain andpost-operative pain, chronic lower back pain, cluster headaches, herpesneuralgia, phantom limb pain, central pain, dental pain,opioid-resistant pain, visceral pain, surgical pain, bone injury pain,pain during labor and delivery, pain resulting from burns, includingsunburn, post partum pain, migraine, angina pain, and genitourinarytract-related pain including cystitis), hepatic disorders (includinghepatitis, alcoholic hepatitis, viral hepatitis, alcoholic cirrhosis, alantitypsin deficiency, autoimmune cirrhosis, cryptogenic cirrhosis,fulminant hepatitis, hepatitis B and C, and steatohepatitis, cysticfibrosis, primary biliary cirrhosis, sclerosing cholangitis and biliaryobstruction), skin and nail disorders (including psoriasis (includingchronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustularpsoriasis and other psoriasis disorders), pemphigus vulgaris,scleroderma, atopic dermatitis (eczema), sarcoidosis, erythema nodosum,hidradenitis suppurative, lichen planus, Sweet's syndrome, sclerodermaand vitiligo), vasculitides (including Behcet's disease), and otherdisorders, such as juvenile rheumatoid arthritis (JRA), endometriosis,prostatitis, choroidal neovascularization, sciatica, Sjogren's syndrome,uveitis, wet macular degeneration, osteoporosis, osteoarthritis, activeaxial spondyloarthritis (active axSpA) and non-radiographic axialspondyloarthritis (nr-axSpA).

As used herein, the term “subject” is intended to include livingorganisms, e.g., prokaryotes and eukaryotes. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In specificembodiments of the invention, the subject is a human.

As used herein, the term “treatment” or “treat” refers to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those already with the disorder, as well asthose in which the disorder is to be prevented.

In one embodiment, the invention provides a method of administering alow AR composition comprising an anti-TNFα antibody, or antigen bindingportion thereof, to a subject such that TNFα activity is inhibited or adisorder in which TNFα activity is detrimental is treated. In oneembodiment, the TNFα is human TNFα and the subject is a human subject.In one embodiment, the anti-TNFα antibody is adalimumab, also referredto as HUMIRA®.

The low AR compositions can be administered by a variety of methodsknown in the art. Exemplary routes/modes of administration includesubcutaneous injection, intravenous injection or infusion. In certainaspects, a low AR compositions may be orally administered. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. In certainembodiments it is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit comprising a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a low AR composition of theinvention is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. With respectto low AR compositions comprising an anti-TNFα antibody, orantigen-binding portion thereof, such as adalimumab, an exemplary doseis 40 mg every other week. In some embodiments, in particular fortreatment of ulcerative colitis or Crohn's disease, an exemplary doseincludes an initial dose (Day 1) of 160 mg (e.g., four 40 mg injectionsin one day or two 40 mg injections per day for two consecutive days), asecond dose two weeks later of 80 mg, and a maintenance dose of 40 mgevery other week beginning two weeks later. Alternatively, for psoriasisfor example, a dosage can include an 80 mg initial dose followed by 40mg every other week starting one week after the initial dose.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

VI. PHARMACEUTICAL FORMULATIONS CONTAINING THE LOW AR COMPOSITIONS OFTHE INVENTION

The present invention further provides preparations and formulationscomprising the low AR compositions of the invention. It should beunderstood that any of the antibodies and antibody fragments describedherein, including antibodies and antibody fragments having any one ormore of the structural and functional features described in detailthroughout the application, may be formulated or prepared as describedbelow. When various formulations are described in this section asincluding an antibody, it is understood that such an antibody may be anantibody or an antibody fragment having any one or more of thecharacteristics of the antibodies and antibody fragments describedherein. In one embodiment, the antibody is an anti-TNFα antibody, orantigen-binding portion thereof.

In certain embodiments, the low AR compositions of the invention may beformulated with a pharmaceutically acceptable carrier as pharmaceutical(therapeutic) compositions, and may be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. The term “pharmaceutically acceptable carrier” meansone or more non-toxic materials that do not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. Such pharmaceutically acceptable preparations may also routinelycontain compatible solid or liquid fillers, diluents or encapsulatingsubstances which are suitable for administration into a human. The term“carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the antibodies of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The low AR compositions of the invention are present in a form known inthe art and acceptable for therapeutic uses. In one embodiment, aformulation of the low AR compositions of the invention is a liquidformulation. In another embodiment, a formulation of the low ARcompositions of the invention is a lyophilized formulation. In a furtherembodiment, a formulation of the low AR compositions of the invention isa reconstituted liquid formulation. In one embodiment, a formulation ofthe low AR compositions of the invention is a stable liquid formulation.In one embodiment, a liquid formulation of the low AR compositions ofthe invention is an aqueous formulation. In another embodiment, theliquid formulation is non-aqueous. In a specific embodiment, a liquidformulation of the low AR compositions of the invention is an aqueousformulation wherein the aqueous carrier is distilled water.

The formulations of the low AR compositions of the invention comprise anantibody in a concentration resulting in a w/v appropriate for a desireddose. The antibody may be present in the formulation at a concentrationof about lmg/ml to about 500 mg/ml, e.g., at a concentration of at least1 mg/ml, at least 5 mg/ml, at least 10 mg/ml, at least 15 mg/ml, atleast 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml,at least 40 mg/ml, at least 45 mg/ml, at least 50 mg/ml, at least 55mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, at least75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least 90 mg/ml, atleast 95 mg/ml, at least 100 mg/ml, at least 105 mg/ml, at least 110mg/ml, at least 115 mg/ml, at least 120 mg/ml, at least 125 mg/ml, atleast 130 mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least 150mg/ml, at least 200 mg/ml, at least 250 mg/ml, or at least 300 mg/ml.

In a specific embodiment, a formulation of the low AR compositions ofthe invention comprises at least about 100 mg/ml, at least about 125mg/ml, at least 130 mg/ml, or at least about 150 mg/ml of an antibody ofthe invention.

In one embodiment, the concentration of antibody, which is included inthe formulation of the invention, is between about 1 mg/ml and about 25mg/ml, between about 1 mg/ml and about 200 mg/ml, between about 25 mg/mland about 200 mg/ml, between about 50 mg/ml and about 200 mg/ml, betweenabout 75 mg/ml and about 200 mg/ml, between about 100 mg/ml and about200 mg/ml, between about 125 mg/ml and about 200 mg/ml, between about150 mg/ml and about 200 mg/ml, between about 25 mg/ml and about 150mg/ml, between about 50 mg/ml and about 150 mg/ml, between about 75mg/ml and about 150 mg/ml, between about 100 mg/ml and about 150 mg/ml,between about 125 mg/ml and about 150 mg/ml, between about 25 mg/ml andabout 125 mg/ml, between about 50 mg/ml and about 125 mg/ml, betweenabout 75 mg/ml and about 125 mg/ml, between about 100 mg/ml and about125 mg/ml, between about 25 mg/ml and about 100 mg/ml, between about 50mg/ml and about 100 mg/ml, between about 75 mg/ml and about 100 mg/ml,between about 25 mg/ml and about 75 mg/ml, between about 50 mg/ml andabout 75 mg/ml, or between about 25 mg/ml and about 50 mg/ml.

In a specific embodiment, a formulation of the low AR compositions ofthe invention comprises between about 90 mg/ml and about 110 mg/ml orbetween about 100 mg/ml and about 210 mg/ml of an antibody.

The formulations of the low AR compositions of the invention comprisingan antibody may further comprise one or more active compounds asnecessary for the particular indication being treated, typically thosewith complementary activities that do not adversely affect each other.Such additional active compound/s is/are suitably present in combinationin amounts that are effective for the purpose intended.

The formulations of the low AR compositions of the invention may beprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers, including, but not limited to buffering agents,saccharides, salts, surfactants, solubilizers, polyols, diluents,binders, stabilizers, salts, lipophilic solvents, amino acids,chelators, preservatives, or the like (Goodman and Gilman's ThePharmacological Basis of Therapeutics, 12^(th) edition, L. Brunton, etal. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.(1999)), in the form of lyophilized formulations or aqueous solutions ata desired final concentration. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as histidine, phosphate, citrate,glycine, acetate and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including trehalose, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g., Zn-protein complexes); and/or non-ionic surfactantssuch as TWEEN, polysorbate 80, PLURONICS™ or polyethylene glycol (PEG).

The buffering agent may be histidine, citrate, phosphate, glycine, oracetate. The saccharide excipient may be trehalose, sucrose, mannitol,maltose or raffinose. The surfactant may be polysorbate 20, polysorbate40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KCl, MgCl₂,or CaCl₂

The formulations of the low AR compositions of the invention may includea buffering or pH adjusting agent to provide improved pH control. Aformulation of the invention may have a pH of between about 3.0 andabout 9.0, between about 4.0 and about 8.0, between about 5.0 and about8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5,between about 5.5 and about 8.0, between about 5.5 and about 7.0, orbetween about 5.5 and about 6.5. In a further embodiment, a formulationof the invention has a pH of about 3.0, about 3.5, about 4.0, about 4.5,about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2,about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In aspecific embodiment, a formulation of the invention has a pH of about6.0. One of skill in the art understands that the pH of a formulationgenerally should not be equal to the isoelectric point of the particularantibody to be used in the formulation.

Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride,or phosphate buffers. In addition, amino acid components can alsofunction in a buffering capacity. Representative amino acid componentswhich may be utilized in the formulations of the invention as bufferingagents include, but are not limited to, glycine and histidine. Incertain embodiments, the buffering agent is chosen from histidine,citrate, phosphate, glycine, and acetate. In a specific embodiment, thebuffering agent is histidine. In another specific embodiment, thebuffering agent is citrate. In yet another specific embodiment, thebuffering agent is glycine. The purity of the buffering agent should beat least 98%, or at least 99%, or at least 99.5%. As used herein, theterm “purity” in the context of histidine and glycine refers to chemicalpurity of histidine or glycine as understood in the art, e.g., asdescribed in The Merck Index, 13^(th) ed., O'Neil et al. ed. (Merck &Co., 2001).

Buffering agents are typically used at concentrations between about 1 mMand about 200 mM or any range or value therein, depending on the desiredionic strength and the buffering capacity required. The usualconcentrations of conventional buffering agents employed in parenteralformulations can be found in: Pharmaceutical Dosage Form: ParenteralMedications, Volume 1, 2^(nd) Edition, Chapter 5, p. 194, De Luca andBoylan, “Formulation of Small Volume Parenterals”, Table 5: Commonlyused additives in Parenteral Products. In one embodiment, the bufferingagent is at a concentration of about 1 mM, or of about 5 mM, or of about10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or ofabout 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, orof about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM,or of about 90 mM, or of about 100 mM. In one embodiment, the bufferingagent is at a concentration of 1 mM, or of 5 mM, or of 10 mM, or of 15mM, or of 20 mM, or of 25 mM, or of 30 mM, or of 35 mM, or of 40 mM, orof 45 mM, or of 50 mM, or of 60 mM, or of 70 mM, or of 80 mM, or of 90mM, or of 100 mM. In a specific embodiment, the buffering agent is at aconcentration of between about 5 mM and about 50 mM. In another specificembodiment, the buffering agent is at a concentration of between 5 mMand 20 mM.

In certain embodiments, the formulation of the low AR compositions ofthe invention comprises histidine as a buffering agent. In oneembodiment the histidine is present in the formulation of the inventionat a concentration of at least about 1 mM, at least about 5 mM, at leastabout 10 mM, at least about 20 mM, at least about 30 mM, at least about40 mM, at least about 50 mM, at least about 75 mM, at least about 100mM, at least about 150 mM, or at least about 200 mM histidine. Inanother embodiment, a formulation of the invention comprises betweenabout 1 mM and about 200 mM, between about 1 mM and about 150 mM,between about 1 mM and about 100 mM, between about 1 mM and about 75 mM,between about 10 mM and about 200 mM, between about 10 mM and about 150mM, between about 10 mM and about 100 mM, between about 10 mM and about75 mM, between about 10 mM and about 50 mM, between about 10 mM andabout 40 mM, between about 10 mM and about 30 mM, between about 20 mMand about 75 mM, between about 20 mM and about 50 mM, between about 20mM and about 40 mM, or between about 20 mM and about 30 mM histidine. Ina further embodiment, the formulation comprises about 1 mM, about 5 mM,about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM,about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In aspecific embodiment, a formulation may comprise about 10 mM, about 25mM, or no histidine.

The formulations of the low AR compositions of the invention maycomprise a carbohydrate excipient. Carbohydrate excipients can act,e.g., as viscosity enhancing agents, stabilizers, bulking agents,solubilizing agents, and/or the like. Carbohydrate excipients aregenerally present at between about 1% to about 99% by weight or volume,e.g., between about 0.1% to about 20%, between about 0.1% to about 15%,between about 0.1% to about 5%, about 1% to about 20%, between about 5%to about 15%, between about 8% to about 10%, between about 10% and about15%, between about 15% and about 20%, between 0.1% to 20%, between 5% to15%, between 8% to 10%, between 10% and 15%, between 15% and 20%,between about 0.1% to about 5%, between about 5% to about 10%, orbetween about 15% to about 20%. In still other specific embodiments, thecarbohydrate excipient is present at 1%, or at 1.5%, or at 2%, or at2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at 20%.

Carbohydrate excipients suitable for use in the formulations of theinvention include, but are not limited to, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.In one embodiment, the carbohydrate excipients for use in the presentinvention are chosen from, sucrose, trehalose, lactose, mannitol, andraffinose. In a specific embodiment, the carbohydrate excipient istrehalose. In another specific embodiment, the carbohydrate excipient ismannitol. In yet another specific embodiment, the carbohydrate excipientis sucrose. In still another specific embodiment, the carbohydrateexcipient is raffinose. The purity of the carbohydrate excipient shouldbe at least 98%, or at least 99%, or at least 99.5%.

In a specific embodiment, the formulations of the low AR compositions ofthe invention may comprise trehalose. In one embodiment, a formulationof the invention comprises at least about 1%, at least about 2%, atleast about 4%, at least about 8%, at least about 20%, at least about30%, or at least about 40% trehalose. In another embodiment, aformulation of the invention comprises between about 1% and about 40%,between about 1% and about 30%, between about 1% and about 20%, betweenabout 2% and about 40%, between about 2% and about 30%, between about 2%and about 20%, between about 4% and about 40%, between about 4% andabout 30%, or between about 4% and about 20% trehalose. In a furtherembodiment, a formulation of the invention comprises about 1%, about 2%,about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about40% trehalose. In a specific embodiment, a formulation of the inventioncomprises about 4%, about 6% or about 15% trehalose.

In certain embodiments, a formulation of the low AR compositions of theinvention comprises an excipient. In a specific embodiment, aformulation of the invention comprises at least one excipient chosenfrom: sugar, salt, surfactant, amino acid, polyol, chelating agent,emulsifier and preservative. In one embodiment, a formulation of theinvention comprises a salt, e.g., a salt selected from: NaCl, KCl,CaCl₂, and MgCl₂. In a specific embodiment, the formulation comprisesNaCl.

A formulation of the low AR compositions of the invention may compriseat least about 10 mM, at least about 25 mM, at least about 50 mM, atleast about 75 mM, at least about 80 mM, at least about 100 mM, at leastabout 125 mM, at least about 150 mM, at least about 175 mM, at leastabout 200 mM, or at least about 300 mM sodium chloride (NaCl). In afurther embodiment, the formulation may comprise between about 10 mM andabout 300 mM, between about 10 mM and about 200 mM, between about 10 mMand about 175 mM, between about 10 mM and about 150 mM, between about 25mM and about 300 mM, between about 25 mM and about 200 mM, between about25 mM and about 175 mM, between about 25 mM and about 150 mM, betweenabout 50 mM and about 300 mM, between about 50 mM and about 200 mM,between about 50 mM and about 175 mM, between about 50 mM and about 150mM, between about 75 mM and about 300 mM, between about 75 mM and about200 mM, between about 75 mM and about 175 mM, between about 75 mM andabout 150 mM, between about 100 mM and about 300 mM, between about 100mM and about 200 mM, between about 100 mM and about 175 mM, or betweenabout 100 mM and about 150 mM sodium chloride. In a further embodiment,the formulation may comprise about 10 mM, about 25 mM, about 50 mM,about 75 mM, about 80 mM, about 100 mM, about 125 mM, about 150 mM,about 175 mM, about 200 mM, or about 300 mM sodium chloride.

A formulation of the low AR compositions of the invention may alsocomprise an amino acid, e.g., lysine, arginine, glycine, histidine or anamino acid salt. The formulation may comprise at least about 1 mM, atleast about 10 mM, at least about 25 mM, at least about 50 mM, at leastabout 100 mM, at least about 150 mM, at least about 200 mM, at leastabout 250 mM, at least about 300 mM, at least about 350 mM, or at leastabout 400 mM of an amino acid. In another embodiment, the formulationmay comprise between about 1 mM and about 100 mM, between about 10 mMand about 150 mM, between about 25 mM and about 250 mM, between about 25mM and about 300 mM, between about 25 mM and about 350 mM, between about25 mM and about 400 mM, between about 50 mM and about 250 mM, betweenabout 50 mM and about 300 mM, between about 50 mM and about 350 mM,between about 50 mM and about 400 mM, between about 100 mM and about 250mM, between about 100 mM and about 300 mM, between about 100 mM andabout 400 mM, between about 150 mM and about 250 mM, between about 150mM and about 300 mM, or between about 150 mM and about 400 mM of anamino acid. In a further embodiment, a formulation of the inventioncomprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about400 mM of an amino acid.

The formulations of the low AR compositions of the invention may furthercomprise a surfactant. The term “surfactant” as used herein refers toorganic substances having amphipathic structures; namely, they arecomposed of groups of opposing solubility tendencies, typically anoil-soluble hydrocarbon chain and a water-soluble ionic group.Surfactants can be classified, depending on the charge of thesurface-active moiety, into anionic, cationic, and nonionic surfactants.Surfactants are often used as wetting, emulsifying, solubilizing, anddispersing agents for various pharmaceutical compositions andpreparations of biological materials. Pharmaceutically acceptablesurfactants like polysorbates (e.g., polysorbates 20 or 80); polyoxamers(e.g., poloxamer 188); Triton; sodium octyl glycoside; lauryl-,myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.), canoptionally be added to the formulations of the invention to reduceaggregation. In one embodiment, a formulation of the invention comprisesPolysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80.Surfactants are particularly useful if a pump or plastic container isused to administer the formulation. The presence of a pharmaceuticallyacceptable surfactant mitigates the propensity for the protein toaggregate. The formulations may comprise a polysorbate which is at aconcentration ranging from between about 0.001% to about 1%, or about0.001% to about 0.1%, or about 0.01% to about 0.1%. In other specificembodiments, the formulations of the invention comprise a polysorbatewhich is at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%,or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or0.015%, or 0.02%.

The formulations of the low AR compositions of the invention mayoptionally further comprise other common excipients and/or additivesincluding, but not limited to, diluents, binders, stabilizers,lipophilic solvents, preservatives, adjuvants, or the like.Pharmaceutically acceptable excipients and/or additives may be used inthe formulations of the invention. Commonly used excipients/additives,such as pharmaceutically acceptable chelators (for example, but notlimited to, EDTA, DTPA or EGTA) can optionally be added to theformulations of the invention to reduce aggregation. These additives areparticularly useful if a pump or plastic container is used to administerthe formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof can optionally beadded to the formulations of the invention at any suitable concentrationsuch as between about 0.001% to about 5%, or any range or value therein.The concentration of preservative used in the formulations of theinvention is a concentration sufficient to yield a microbial effect.Such concentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in theformulations of the invention include, for example, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,lipids such as phospholipids or fatty acids, steroids such ascholesterol, protein excipients such as serum albumin (human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,salt-forming counterions such as sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 21^(st) ed.,Lippincott Williams & Wilkins, (2005), and in the “Physician's DeskReference”, 60^(th) ed., Medical Economics, Montvale, N.J. (2005).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofan antibody, as well known those in the art or as described herein.

In one embodiment, the low AR compositions of the invention areformulated with the same or similar excipients and buffers as arepresent in the commercial adalimumab (HUMIRA®) formulation, as describedin the “Highlights of HUMIRA® Prescribing Information” for HUMIRA®(adalimumab) Injection (Revised January 2008) the entire contents ofwhich are expressly incorporated herein by reference. For example, eachprefilled syringe of HUMIRA®, which is administered subcutaneously,delivers 0.8 mL (40 mg) of drug product to the subject. Each 0.8 mL ofHUMIRA® contains 40 mg adalimumab, 4.93 mg sodium chloride, 0.69 mgmonobasic sodium phosphate dihydrate, 1.22 mg dibasic sodium phosphatedihydrate, 0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6mg mannitol, 0.8 mg polysorbate 80, and water for Injection, USP. Sodiumhydroxide is added as necessary to adjust pH.

It will be understood by one skilled in the art that the formulations ofthe low AR compositions of the invention may be isotonic with humanblood, wherein the formulations of the invention have essentially thesame osmotic pressure as human blood. Such isotonic formulations willgenerally have an osmotic pressure from about 250 mOSm to about 350mOSm. Isotonicity can be measured by, for example, using a vaporpressure or ice-freezing type osmometer. Tonicity of a formulation isadjusted by the use of tonicity modifiers. “Tonicity modifiers” arethose pharmaceutically acceptable inert substances that can be added tothe formulation to provide an isotonity of the formulation. Tonicitymodifiers suitable for this invention include, but are not limited to,saccharides, salts and amino acids.

In certain embodiments, the formulations of the low AR compositions ofthe invention have an osmotic pressure from about 100 mOSm to about 1200mOSm, or from about 200 mOSm to about 1000 mOSm, or from about 200 mOSmto about 800 mOSm, or from about 200 mOSm to about 600 mOSm, or fromabout 250 mOSm to about 500 mOSm, or from about 250 mOSm to about 400mOSm, or from about 250 mOSm to about 350 mOSm.

The concentration of any one component or any combination of variouscomponents, of the formulations of the low AR compositions of theinvention is adjusted to achieve the desired tonicity of the finalformulation. For example, the ratio of the carbohydrate excipient toantibody may be adjusted according to methods known in the art (e.g.,U.S. Pat. No. 6,685,940). In certain embodiments, the molar ratio of thecarbohydrate excipient to antibody may be from about 100 moles to about1000 moles of carbohydrate excipient to about 1 mole of antibody, orfrom about 200 moles to about 6000 moles of carbohydrate excipient toabout 1 mole of antibody, or from about 100 moles to about 510 moles ofcarbohydrate excipient to about 1 mole of antibody, or from about 100moles to about 600 moles of carbohydrate excipient to about 1 mole ofantibody.

The desired isotonicity of the final formulation may also be achieved byadjusting the salt concentration of the formulations. Pharmaceuticallyacceptable salts and those suitable for this invention as tonicitymodifiers include, but are not limited to, sodium chloride, sodiumsuccinate, sodium sulfate, potassuim chloride, magnesium chloride,magnesium sulfate, and calcium chloride. In specific embodiments,formulations of the invention comprise NaCl, MgCl₂, and/or CaCl₂. In oneembodiment, concentration of NaCl is between about 75 mM and about 150mM. In another embodiment, concentration of MgCl₂ is between about 1 mMand about 100 mM. Pharmaceutically acceptable amino acids includingthose suitable for this invention as tonicity modifiers include, but arenot limited to, proline, alanine, L-arginine, asparagine, L-asparticacid, glycine, serine, lysine, and histidine.

In one embodiment the formulations of the low AR compositions of theinvention are pyrogen-free formulations which are substantially free ofendotoxins and/or related pyrogenic substances. Endotoxins includetoxins that are confined inside a microorganism and are released onlywhen the microorganisms are broken down or die. Pyrogenic substancesalso include fever-inducing, thermostable substances (glycoproteins)from the outer membrane of bacteria and other microorganisms. Both ofthese substances can cause fever, hypotension and shock if administeredto humans. Due to the potential harmful effects, even low amounts ofendotoxins must be removed from intravenously administeredpharmaceutical drug solutions. The Food & Drug Administration (“FDA”)has set an upper limit of 5 endotoxin units (EU) per dose per kilogrambody weight in a single one hour period for intravenous drugapplications (The United States Pharmacopeial Convention, PharmacopeialForum 26 (1):223 (2000)). When therapeutic proteins are administered inamounts of several hundred or thousand milligrams per kilogram bodyweight, as can be the case with antibodies, even trace amounts ofharmful and dangerous endotoxin must be removed. In certain specificembodiments, the endotoxin and pyrogen levels in the composition areless then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or lessthen 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.

When used for in vivo administration, the formulations of the low ARcompositions of the invention should be sterile. The formulations of theinvention may be sterilized by various sterilization methods, includingsterile filtration, radiation, etc. In one embodiment, the antibodyformulation is filter-sterilized with a presterilized 0.22-micronfilter. Sterile compositions for injection can be formulated accordingto conventional pharmaceutical practice as described in “Remington: TheScience & Practice of Pharmacy”, 21^(st) ed., Lippincott Williams &Wilkins, (2005). Formulations comprising antibodies, such as thosedisclosed herein, ordinarily will be stored in lyophilized form or insolution. It is contemplated that sterile compositions comprisingantibodies are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having an adapter thatallows retrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle. In one embodiment, a composition of theinvention is provided as a pre-filled syringe.

In one embodiment, a formulation of the low AR compositions of theinvention is a lyophilized formulation. The term “lyophilized” or“freeze-dried” includes a state of a substance that has been subjectedto a drying procedure such as lyophilization, where at least 50% ofmoisture has been removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Bulking agents known to theart include, for example, carbohydrates, including simple sugars such asdextrose, ribose, fructose and the like, alcohol sugars such asmannitol, inositol and sorbitol, disaccharides including trehalose,sucrose and lactose, naturally occurring polymers such as starch,dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serumalbumin), glycogen, and synthetic monomers and polymers.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest (such as an antibody of the invention), significantly preventsor reduces chemical and/or physical instability of the protein uponlyophilization and subsequent storage. Lyoprotectants include, but arenot limited to, sugars and their corresponding sugar alcohols; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher molecular weight sugar alcohols, e.g., glycerin,dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, andmannitol; propylene glycol; polyethylene glycol; PLURONICS™; andcombinations thereof. Additional examples of lyoprotectants include, butare not limited to, glycerin and gelatin, and the sugars mellibiose,melezitose, raffinose, mannotriose and stachyose. Examples of reducingsugars include, but are not limited to, glucose, maltose, lactose,maltulose, iso-maltulose and lactulose. Examples of non-reducing sugarsinclude, but are not limited to, non-reducing glycosides of polyhydroxycompounds selected from sugar alcohols and other straight chainpolyalcohols. Examples of sugar alcohols include, but are not limitedto, monoglycosides, compounds obtained by reduction of disaccharidessuch as lactose, maltose, lactulose and maltulose. The glycosidic sidegroup can be either glucosidic or galactosidic. Additional examples ofsugar alcohols include, but are not limited to, glucitol, maltitol,lactitol and iso-maltulose. In specific embodiments, trehalose orsucrose is used as a lyoprotectant.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

In one embodiment, the molar ratio of a lyoprotectant (e.g., trehalose)and antibody molecules of a formulation of the invention is at leastabout 10, at least about 50, at least about 100, at least about 200, orat least about 300. In another embodiment, the molar ratio of alyoprotectant (e.g., trehalose) and antibody molecules of a formulationof the invention is about 1, is about 2, is about 5, is about 10, about50, about 100, about 200, or about 300.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized antibody formulation in a diluent such that theantibody is dispersed in the reconstituted formulation. Thereconstituted formulation is suitable for administration (e.g.,parenteral administration) to a patient to be treated with the antibodyand, in certain embodiments of the invention, may be one which issuitable for intravenous administration.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. In some embodiments,diluents include, but are not limited to, sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution. In an alternative embodiment, diluents can includeaqueous solutions of salts and/or buffers.

In certain embodiments, a formulation of the low AR compositions of theinvention is a lyophilized formulation comprising an antibody of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of said antibodymay be recovered from a vial upon shaking said vial for 4 hours at aspeed of 400 shakes per minute wherein the vial is filled to half of itsvolume with the formulation. In another embodiment, a formulation of theinvention is a lyophilized formulation comprising an antibody of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of the antibody maybe recovered from a vial upon subjecting the formulation to threefreeze/thaw cycles wherein the vial is filled to half of its volume withsaid formulation. In a further embodiment, a formulation of theinvention is a lyophilized formulation comprising an antibody of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of the antibody maybe recovered by reconstituting a lyophilized cake generated from saidformulation.

In one embodiment, a reconstituted liquid formulation may comprise anantibody at the same concentration as the pre-lyophilized liquidformulation.

In another embodiment, a reconstituted liquid formulation may comprisean antibody at a higher concentration than the pre-lyophilized liquidformulation, e.g., about 2 fold, about 3 fold, about 4 fold, about 5fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about10 fold higher concentration of an antibody than the pre-lyophilizedliquid formulation.

In yet another embodiment, a reconstituted liquid formulation maycomprise an antibody of the invention at a lower concentration than thepre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold,about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold,about 9 fold or about 10 fold lower concentration of an antibody thanthe pre-lyophilized liquid formulation.

The pharmaceutical formulations of the low AR compositions of theinvention are typically stable formulations, e.g., stable at roomtemperature.

The terms “stability” and “stable” as used herein in the context of aformulation comprising an antibody of the invention refer to theresistance of the antibody in the formulation to aggregation,degradation or fragmentation under given manufacture, preparation,transportation and storage conditions. The “stable” formulations of theinvention retain biological activity under given manufacture,preparation, transportation and storage conditions. The stability of theantibody can be assessed by degrees of aggregation, degradation orfragmentation, as measured by HPSEC, static light scattering (SLS),Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD),urea unfolding techniques, intrinsic tryptophan fluorescence,differential scanning calorimetry, and/or ANS binding techniques,compared to a reference formulation. For example, a referenceformulation may be a reference standard frozen at −70° C. consisting of10 mg/ml of an antibody of the invention in PBS.

Therapeutic formulations of the low AR compositions of the invention maybe formulated for a particular dosage. Dosage regimens may be adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the antibody and theparticular therapeutic effect to be achieved, and (b) the limitationsinherent in the art of compounding such an antibody for the treatment ofsensitivity in individuals.

Therapeutic compositions of the low AR compositions of the invention canbe formulated for particular routes of administration, such as oral,nasal, pulmonary, topical (including buccal and sublingual), rectal,vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods known in the art of pharmacy. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will vary depending upon the subject being treated, and theparticular mode of administration. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the composition which produces atherapeutic effect. By way of example, in certain embodiments, theantibodies (including antibody fragments) are formulated for intravenousadministration. In certain other embodiments, the antibodies (includingantibody fragments) are formulated for local delivery to thecardiovascular system, for example, via catheter, stent, wire,intramyocardial delivery, intrapericardial delivery, or intraendocardialdelivery.

Formulations of the low AR compositions of the invention which aresuitable for topical or transdermal administration include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required (U.S. Pat. Nos. 7,378,110;7,258,873; 7,135,180; 7,923,029; and US Publication No. 2004-0042972 and2004-0042971).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the low AR compositions of the invention may be variedso as to obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration, without being toxic to thepatient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

In certain embodiments, antibodies of the invention can be formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention can cross the BBB (if desired),they can be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,(1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G.Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)Antimicrob. Agents Chemother. 39:180); surfactant Protein A receptor(Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species ofwhich may comprise the formulations of the invention, as well ascomponents of the invented molecules; p120 (Schreier et al. (1994) J.Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBSLett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.In one embodiment of the invention, the therapeutic compounds of theinvention are formulated in liposomes; in another embodiment, theliposomes include a targeting moiety. In another embodiment, thetherapeutic compounds in the liposomes are delivered by bolus injectionto a site proximal to the desired area. When administered in thismanner, the composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and may be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. Additionally oralternatively, the antibodies of the invention may be delivered locallyto the brain to mitigate the risk that the blood brain barrier slowseffective delivery.

In certain embodiments, the low AR compositions of the invention may beadministered with medical devices known in the art. For example, incertain embodiments an antibody or antibody fragment is administeredlocally via a catheter, stent, wire, or the like. For example, in oneembodiment, a therapeutic composition of the invention can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

The efficient dosages and the dosage regimens for the low ARcompositions of the invention depend on the disease or condition to betreated and can be determined by the persons skilled in the art. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

VII. ALTERNATIVE FORMULATIONS CONTAINING THE LOW AR COMPOSITIONS OF THEINVENTION

a. Aqueous Formulations

The invention also provides a low AR composition formulated as anaqueous formulation comprising a protein and water, as described in U.S.Pat. No. 8,420,081 and PCT Publication No. WO2012/065072, the contentsof which are hereby incorporated by reference. In these aqueousformulations, the protein is stable without the need for additionalagents. This aqueous formulation has a number of advantages overconventional formulations in the art, including stability of the proteinin water without the requirement for additional excipients, increasedconcentrations of protein without the need for additional excipients tomaintain solubility of the protein, and low osmolality. These also haveadvantageous storage properties, as the proteins in the formulationremain stable during storage, e.g., stored as a liquid form for morethan 3 months at 7° C. or freeze/thaw conditions, even at high proteinconcentrations and repeated freeze/thaw processing steps. In oneembodiment, formulations described herein include high concentrations ofproteins such that the aqueous formulation does not show significantopalescence, aggregation, or precipitation.

In one embodiment, an aqueous low AR composition comprising a protein,e.g., an antibody, e.g., an anti-TNFα antibody or antigen biding portionthereof, and water is provided, wherein the formulation has certaincharacteristics, such as, but not limited to, low conductivity, e.g., aconductivity of less than about 2.5 mS/cm, a protein concentration of atleast about 10 μg/mL, an osmolality of no more than about 30 mOsmol/kg,and/or the protein has a molecular weight (Mw) greater than about 47kDa. In one embodiment, the formulation has improved stability, such as,but not limited to, stability in a liquid form for an extended time(e.g., at least about 3 months or at least about 12 months) or stabilitythrough at least one freeze/thaw cycle (if not more freeze/thaw cycles).In one embodiment, the formulation is stable for at least about 3 monthsin a form selected from the group consisting of frozen, lyophilized, orspray-dried.

In one embodiment, the formulation has a low conductivity, including,for example, a conductivity of less than about 2.5 mS/cm, a conductivityof less than about 2 mS/cm, a conductivity of less than about 1.5 mS/cm,a conductivity of less than about 1 mS/cm, or a conductivity of lessthan about 0.5 mS/cm.

In another embodiment, low AR compositions included in the formulationhave a given concentration, including, for example, a concentration ofat least about 1 mg/mL, at least about 10 mg/mL, at least about 50mg/mL, at least about 100 mg/mL, at least about 150 mg/mL, at leastabout 200 mg/mL, or greater than about 200 mg/mL. In another embodiment,the formulation of the invention has an osmolality of no more than about15 mOsmol/kg.

The aqueous formulations described herein do not rely on standardexcipients, e.g., a tonicity modifier, a stabilizing agent, asurfactant, an anti-oxidant, a cryoprotectant, a bulking agent, alyroprotectant, a basic component, and an acidic component. In otherembodiments of the invention, the formulation contains water, one ormore proteins, and no ionic excipients (e.g., salts, free amino acids).

In certain embodiments, the aqueous formulation as described hereincomprise a low AR composition comprising a protein concentration of atleast 50 mg/mL and water, wherein the formulation has an osmolality ofno more than 30 mOsmol/kg. Lower limits of osmolality of the aqueousformulation are also encompassed by the invention. In one embodiment theosmolality of the aqueous formulation is no more than 15 mOsmol/kg. Theaqueous formulation of the invention may have an osmolality of less than30 mOsmol/kg, and also have a high protein concentration, e.g., theconcentration of the protein is at least 100 mg/mL, and may be as muchas 200 mg/mL or greater. Ranges intermediate to the above recitedconcentrations and osmolality units are also intended to be part of thisinvention. In addition, ranges of values using a combination of any ofthe above recited values as upper and/or lower limits are intended to beincluded.

The concentration of the aqueous formulation as described herein is notlimited by the protein size and the formulation may include any sizerange of proteins. Included within the scope of the invention is anaqueous formulation comprising at least 40 mg/mL and as much as 200mg/mL or more of a protein, for example, 40 mg/mL, 65 mg/mL, 130 mg/mL,or 195 mg/ml, which may range in size from 5 kDa to 150 kDa or more. Inone embodiment, the protein in the formulation of the invention is atleast about 15 kD in size, at least about 20 kD in size; at least about47 kD in size; at least about 60 kD in size; at least about 80 kD insize; at least about 100 kD in size; at least about 120 kD in size; atleast about 140 kD in size; at least about 160 kD in size; or greaterthan about 160 kD in size. Ranges intermediate to the above recitedsizes are also intended to be part of this invention. In addition,ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included.

The aqueous formulation as described herein may be characterized by thehydrodynamic diameter (D_(h)) of the proteins in solution. Thehydrodynamic diameter of the protein in solution may be measured usingdynamic light scattering (DLS), which is an established analyticalmethod for determining the D_(h) of proteins. Typical values formonoclonal antibodies, e.g., IgG, are about 10 nm. Low-ionicformulations may be characterized in that the D_(h) of the proteins arenotably lower than protein formulations comprising ionic excipients. Ithas been discovered that the D_(h) values of antibodies in aqueousformulations made using the disfiltration/ultrafilteration (DF/UF)process, as described in U.S. Pat. No. 8,420,081 and PCT Publication No.WO2012/065072, using pure water as an exchange medium, are notably lowerthan the D_(h) of antibodies in conventional formulations independent ofprotein concentration. In one embodiment, antibodies in the aqueousformulation as described herein have a D_(h) of less than 4 nm, or lessthan 3 nm.

In one embodiment, the D_(h) of the protein in the aqueous formulationis smaller relative to the D_(h) of the same protein in a bufferedsolution, irrespective of protein concentration. Thus, in certainembodiments, protein in an aqueous formulation made in accordance withthe methods described herein, will have a D_(h) which is at least 25%less than the D_(h) of the protein in a buffered solution at the samegiven concentration. Examples of buffered solutions include, but are notlimited to phosphate buffered saline (PBS). In certain embodiments,proteins in the aqueous formulation of the invention have a D_(h) thatis at least 50% less than the D_(h) of the protein in PBS in at thegiven concentration; at least 60% less than the D_(h) of the protein inPBS at the given concentration; at least 70% less than the D_(h) of theprotein in PBS at the given concentration; or more than 70% less thanthe D_(h) of the protein in PBS at the given concentration. Rangesintermediate to the above recited percentages are also intended to bepart of this invention, e.g., about 55%, 56%, 57%, 64%, 68%, and soforth. In addition, ranges of values using a combination of any of theabove recited values as upper and/or lower limits are intended to beincluded, e.g., about 50% to about 80%.

In one aspect, the aqueous formulation includes the protein at a dosageof about 0.01 mg/kg-10 mg/kg. In another aspect, the dosages of theprotein include approximately 1 mg/kg administered every other week, orapproximately 0.3 mg/kg administered weekly. A skilled practitioner canascertain the proper dosage and regime for administering to a subject.

b. “Solid Unit” Formulations

The present invention also provides a low AR composition of theinvention formulated as a stable composition of a protein, e.g.,particularly a therapeutic protein such as an antibody, or antigenbinding portion thereof, and a stabilizer, referred to herein as solidunits. These formulations are described, for example, in U.S.Provisional Patent Application 61/893,123, entitled “Stable SolidProtein Compositions and Methods of Making Same”, Attorney Docket Number117813-31001, filed on Oct. 18, 2013, the entire contents of which areexpressly incorporated herein by reference.

Specifically, it has been discovered that despite having a highproportion of sugar relative to the protein, the solid units of theinvention maintain structural rigidity and resist changes in shapeand/or volume when stored under ambient conditions, e.g., roomtemperature and humidity, for extended periods of time. The solid unitsof the invention remain free-flowing and are able to maintain long-termphysical and chemical stability of the protein without significantdegradation and/or aggregate formation. The solid units of the inventionhave many advantages over the art, including that they can be formulatedfor oral delivery and are easily reconstituted in a diluent, such aswater. Because the solid units are readily dissolved, they may be usedin dual chamber delivery devices and may be prepared directly in adevice for patient use.

As used herein, the term “solid unit,” refers to a composition which issuitable for pharmaceutical administration and comprises a protein,e.g., an antibody or peptide, and a stabilizer, e.g., a sugar. The solidunit has a structural rigidity and resistance to changes in shape and/orvolume. In a preferred embodiment, the solid unit is obtained bylyophilizing a pharmaceutical formulation of a therapeutic protein. Thesolid unit may be any shape, e.g., geometric shape, including, but notlimited to, a sphere, a cube, a pyramid, a hemisphere, a cylinder, ateardrop, and so forth, including irregularly shaped units. In oneembodiment, the solid unit has a volume ranging from about 1 ml to about20 ml. In one embodiment, the solid unit is not obtained using spraydrying techniques, e.g., the solid unit is not a powder or granule.

As used herein, the phrase “a plurality of solid units” refers to acollection or population of solid units, wherein the collectioncomprises two or more solid units having a substantially uniform shape,e.g., sphere, and/or volume distribution. In one embodiment, theplurality of solid units is free-flowing.

VIII. KITS AND ARTICLES OF MANUFACTURE COMPRISING THE LOW ARCOMPOSITIONS OF THE INVENTION

Also within the scope of the present invention are kits comprising thelow AR compositions of the invention and instructions for use. The term“kit” as used herein refers to a packaged product comprising componentswith which to administer the antibody, or antigen-binding portionthereof, of the invention for treatment of a disease or disorder. Thekit may comprise a box or container that holds the components of thekit. The box or container is affixed with a label or a Food and DrugAdministration approved protocol. The box or container holds componentsof the invention which may be contained within plastic, polyethylene,polypropylene, ethylene, or propylene vessels. The vessels can becapped-tubes or bottles. The kit can also include instructions foradministering an antibody of the invention.

The kit can further contain one more additional reagents, such as animmunosuppressive reagent, a cytotoxic agent or a radiotoxic agent orone or more additional antibodies of the invention (e.g., an antibodyhaving a complementary activity which binds to an epitope in the TNFαantigen distinct from a first anti-TNFα antibody). Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a liquid formulation or lyophilizedformulation of an antibody or antibody fragment thereof of theinvention. In one embodiment, a container filled with a liquidformulation of the invention is a pre-filled syringe. In a specificembodiment, the formulations of the invention are formulated in singledose vials as a sterile liquid. For example, the formulations may besupplied in 3 cc USP Type I borosilicate amber vials (WestPharmaceutical Services—Part No. 6800-0675) with a target volume of 1.2mL. Optionally associated with such container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

In one embodiment, a container filled with a liquid formulation of theinvention is a pre-filled syringe. Any pre-filled syringe known to oneof skill in the art may be used in combination with a liquid formulationof the invention. Pre-filled syringes that may be used are described in,for example, but not limited to, PCT Publications WO05032627,WO08094984, WO9945985, WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat.No. 5,607,400, U.S. Pat. No. 5,893,842, U.S. Pat. No. 7,081,107, U.S.Pat. No. 7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat. No. 6,807,797,U.S. Pat. No. 6,142,976, U.S. Pat. No. 5,899,889, U.S. Pat. No.7,699,811, U.S. Pat. No. 7,540,382, U.S. Pat. No. 7,998,120, U.S. Pat.No. 7,645,267, and US Patent Publication No. US20050075611. Pre-filledsyringes may be made of various materials. In one embodiment apre-filled syringe is a glass syringe. In another embodiment apre-filled syringe is a plastic syringe. One of skill in the artunderstands that the nature and/or quality of the materials used formanufacturing the syringe may influence the stability of a proteinformulation stored in the syringe. For example, it is understood thatsilicon based lubricants deposited on the inside surface of the syringechamber may affect particle formation in the protein formulation. In oneembodiment, a pre-filled syringe comprises a silicone based lubricant.In one embodiment, a pre-filled syringe comprises baked on silicone. Inanother embodiment, a pre-filled syringe is free from silicone basedlubricants. One of skill in the art also understands that small amountsof contaminating elements leaching into the formulation from the syringebarrel, syringe tip cap, plunger or stopper may also influence stabilityof the formulation. For example, it is understood that tungstenintroduced during the manufacturing process may adversely affectformulation stability. In one embodiment, a pre-filled syringe maycomprise tungsten at a level above 500 ppb. In another embodiment, apre-filled syringe is a low tungsten syringe. In another embodiment, apre-filled syringe may comprise tungsten at a level between about 500ppb and about 10 ppb, between about 400 ppb and about 10 ppb, betweenabout 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb,between about 100 ppb and about 10 ppb, between about 50 ppb and about10 ppb, between about 25 ppb and about 10 ppb.

In certain embodiments, kits comprising antibodies of the invention arealso provided that are useful for various purposes, e.g., research anddiagnostic including for purification or immunoprecipitation of proteinof interest from cells, detection of the protein of interest in vitro orin vivo. For isolation and purification of a protein of interest, thekit may contain an antibody coupled to beads (e.g., sepharose beads).Kits may be provided which contain the antibodies for detection andquantitation of a protein of interest in vitro, e.g., in an ELISA or aWestern blot. As with the article of manufacture, the kit comprises acontainer and a label or package insert on or associated with thecontainer. The container holds a composition comprising at least oneantibody of the invention. Additional containers may be included thatcontain, e.g., diluents and buffers, control antibodies. The label orpackage insert may provide a description of the composition as well asinstructions for the intended in vitro or diagnostic use.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial, pre-filled syringe or other container that ishermetically sealed. In one embodiment, the unit dosage form is providedas a sterile particulate free solution comprising an antibody that issuitable for parenteral administration. In another embodiment, the unitdosage form is provided as a sterile lyophilized powder comprising anantibody that is suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses sterile solutions suitable for each deliveryroute. The invention further encompasses sterile lyophilized powdersthat are suitable for reconstitution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the pharmaceutical. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information. Specifically, theinvention provides an article of manufacture comprising packagingmaterial, such as a box, bottle, tube, vial, container, pre-filledsyringe, sprayer, insufflator, intravenous (i.v.) bag, envelope and thelike; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises a liquid formulation containing an antibody. Thepackaging material includes instruction means which indicate how thatsaid antibody can be used to prevent, treat and/or manage one or moresymptoms associated with a disease or disorder.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references, including literature references, issued patents,and published patent applications, as cited throughout this applicationare hereby expressly incorporated herein by reference. It should furtherbe understood that the contents of all the figures and tables attachedhereto are expressly incorporated herein by reference. The entirecontents of the following applications are also expressly incorporatedherein by reference: U.S. Provisional Patent Application 61/893,123,entitled “STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OF MAKING SAME”,Attorney Docket Number 117813-31001, filed on Oct. 18, 2013; U.S.Provisional Application Ser. No. 61/892,833, entitled “LOW ACIDICSPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME USINGDISPLACEMENT CHROMATOGRAPHY”, Attorney Docket Number 117813-73602, filedon Oct. 18, 2013; U.S. Provisional Patent Application 61/892,710,entitled “MUTATED ANTI-TNFα ANTIBODIES AND METHODS OF THEIR USE”,Attorney Docket Number 117813-73802, filed on Oct. 18, 2013; U.S.Provisional Patent Application 61/893,068, entitled “LOW ACIDIC SPECIESCOMPOSITIONS AND METHODS FOR PRODUCING THE SAME”, Attorney Docket Number117813-73901, filed on Oct. 18, 2013; U.S. Provisional PatentApplication 61/893,088, entitled “MODULATED LYSINE VARIANT SPECIES ANDMETHODS FOR PRODUCING AND USING THE SAME”, Attorney Docket Number117813-74101, filed on Oct. 18, 2013; and U.S. Provisional PatentApplication 61/893,131, entitled “PURIFICATION OF PROTEINS USINGHYDROPHOBIC INTERACTION CHROMATOGRAPHY”, Attorney Docket Number117813-74301, filed on Oct. 18, 2013.

IX. EXAMPLES

Three antibodies were used in connection with the studies outlined below(Examples 1-8). Adalimumab antibody was generated from cell cultureprocessed using chemical defined medium (CDM) and purified by a 4.4 cm(id.)×˜20 cm (L) MabSelect SuRe Protein A column. mAb X bulk drugsubstance was obtained from a three-step large scale purificationprocess. mAb Y antibody was generated from a large scale manufacturingprocess and purified by a MabSelect SuRe Protein A column. AdalimumabProtein A eluate was in a buffer of 20 mM acetic acid at pH˜4.2. The mAbX was in a buffer containing ˜15 mM histidine, pH˜6. The mAb Y was in abuffer containing ˜10 mM sodium formate, pH˜4.2. Each mAb feed wasconditioned to the targeted pH, conductivity and concentration prior tothe displacement chromatography experiment.

The cationic displacers, Expell SP1™ and protamine sulfate (from salmonsperm), were purchased from SACHEM Chemical Company and Sigma Aldrich,respectively.

Poros XS CEX resin (Life Technologies) was packed in a 0.66 cm×˜25 cmcolumn. The column was equilibrated with a 140 mM Tris/Acetate buffer ora 30 mM MES, 10 mM NaCl buffer at the targeted pH and conductivity(Table 1). After equilibration, the column was loaded with eachpre-conditioned feed at a resin loading level of ˜40 g/L followed by a 2CV of equilibration buffer wash. The displacing buffer, which consistsof defined concentration of Expell SP1™ or protamine sulfate in theequilibration buffer, was flowed through the column to initiate thedisplacement process. In standard one-step displacement wash process,this step was continued for at least 30 CV at a flow rate correspondingto 15 to 22 min residence time (RT) before column regeneration andcleaning with a caustic solution consisting of 0.5 N NaOH and 0.5 M KCl.Alternatively, the displacement wash step comprised two displacementbuffers each flowing for defined volumes, or a linear gradient flow fromlow to high concentration displacer buffer. Sample fractions werecollected at every 0.5 or 1 CV for protein concentration and qualityanalysis. The specific processing conditions are detailed in Tables 1and 2.

Capto MMC resin (GE Healthcare) was packed in a 0.66 cm×˜30 cm column.The column was equilibrated with a 140 mM Tris/Acetate buffer at thetargeted pH and conductivity (Table 3). After equilibration, the columnwas loaded with each pre-conditioned feed at a resin loading level about34 to 40 g/L followed by a 2 CV equilibration buffer wash. Thedisplacing buffer, which consists of defined concentration of protaminesulfate in the equilibration buffer, was flowed through the column toinitiate the displacement process. This step was continued for 30 CV ata flow rate corresponding to ˜22 min RT before column regeneration andcleaning. Sample fractions were collected at every 0.5 or 1 CV forprotein concentration and quality analysis. The specific processingconditions are detailed in Table 3.

The levels of acidic species and other charge variants in theAdalimumab, mAb X and mAb Y samples were quantified using the respectivequalified CEX-HPLC method. For Adalimumab, a 4 mm×250 mm analyticalDionex ProPac WCX-10 column (Dionex, CA) was used along with a ShimazhuHPLC system. The mobile phases were 10 mM Sodium Phosphate dibasic pH7.5 buffer (Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mMSodium Chloride pH 5.5 buffer (Mobile phase B). A binary gradient (6% B:0 min; 6-16% B: 0-20 min; 16-100% B: 20-22 min; 100% B: 22-26 min;100-6% B: 26-28 min; 6% B: 28-35 min) was used with detection at 280 nm.Quantitation was based on the relative area percentage of detectedpeaks. The peaks that elute at residence time less than ˜7 min weretogether represented as the acidic peaks or AR region.

For mAb X, a 4 mm×250 mm analytical Dionex ProPac WCX-10 column (Dionex,CA) was used along with a Shimazhu HPLC system. The mobile phases were20 mM MES, pH 6.5 buffer (Mobile phase A) and 20 mM MES, 500 mM NaCl, pH6.5 buffer (Mobile phase B). A binary gradient (10% B: 0 min; 10-28% B:1-46 min; 28-100% B: 46-47 min; 100% B: 47-52 min; 100-10% B: 52-53 min;10% B: 53-58 min) was used with detection at 280 nm. Quantitation wasbased on the relative area percentage of detected peaks. All peakseluting prior to the Main Isoform peak were summed as the acidic region,and all peaks eluting after the Main peak were summed as the basicregion.

For mAb Y, a 4 mm×250 mm Dionex ProPac analytical WCX-10 column (Dionex,CA) was used on a Shimazhu HPLC system. The mobile phases were 20 mMMES, pH 6.2 (Mobile phase A) and 20 mM MES, 250 mM NaCl, pH 6.2 (Mobilephase B). A binary gradient (1% B: 0-1 min; 1-25% B: 1-46 min; 25-100%B: 46-47 min; 100% B: 47-52 min; 100-1% B: 52-53 min; 1% B: 53-60 min)was used with detection at 280 nm. Column temperature was set at 35° C.Quantitation was based on the relative area percentage of detectedpeaks. All peaks eluting prior to the Main Isoform peak (but after 2 minretention time) were summed as the acidic region, and all peaks elutingafter the Main peak were summed as the basic region.

The levels of aggregates, monomer and fragments in eluate samples weremeasured using a SEC method for each molecule. For Adalimumab and mAb Y,a TSK-gel G3000SWxL, 5 μm, 125 Å, 7.8×300 mm column (Tosoh Bioscience)was used while a TSK-gel Super SW3000, 4 μm, 250 Å, 4.6×300 mm column(Tosoh Bioscience) was used for mAb X along with an Agilent or aShimazhu HPLC system. For Adalimumab and mAb X, injections were madeunder isocratic elution conditions using a mobile phase consisting of100 mM sodium sulfate and 100 mM sodium phosphate at pH 6.8, anddetected with UV absorbance at 214 nm. For mAb Y, the mobile phaseconsists of 1×PBS at pH 7.4, and elution profile detected with UVabsorbance at 280 nm. Quantification is based on the relative area ofdetected peaks.

An HCP ELISA assay was used to determine the HCP levels in varioussamples and feeds for all three mAbs.

TABLE 1 Processing conditions for Poros XS one-step displacementchromatography Equilibration/Wash/Displacing Displacer buffer Conc.Buffer Conductivity Loading Molecule Displacer (mM) System pH (mS/cm)Conditions Regeneration Adalimumab Expell 0.5-3 Tris/Acetate 6.7-7.85.4-6.6 pH ~7.5, ~6 2M NaCl SP1 mS/cm  2-5 MES/NaCl   6.1   2.1 pH 6.1,~2 0.2M acetic acid & mS/cm 1M KCl Protamine 0.25-2  Tris/Acetate6.5-7.5 5.6-6.6 pH 7.5, 5.4- 2M NaCl, 6M Sulfate 6.3 mS/cm Guanidine HClmAb X Expell 0.5-2 Tris/Acetate 6 6.2-6.5 pH 6, ~6 2M NaCl SP1 mS/cmProtamine  0.25-0.5 Tris/Acetate 6 6.0-6.5 pH 6, 5.6-6.5 2M NaCl, 6MSulfate mS/cm Guanidine HCl mAb Y Expell 0.5-1 Tris/Acetate 5 ~6 pH 5,6.2 2M NaCl SP1 mS/cm

TABLE 2 Processing conditions for Poros XS two-step or linear gradientdisplacement chromatography Displacer Equilibration/Wash/Displacingbuffer Displacement Concentration Buffer Conductivity Loading MoleculeDisplacer Method (mM) System pH (mS/cm) Conditions RegenerationAdalimumab Expell Two- (1): 0.5 mM, Tris/ 7 ~6 pH 7.5, 2M NaCl SP1 step25CV; Acetate 6.1 (2): 2 mM, mS/cm 20CV Protamine Two- (1): Tris/ 7.55.5 pH 7.5, 2M NaCl, Sulfate step 0.25 mM, Acetate 6.1 6M 10CV; mS/cmGuanidine (2): 2 mM, HCl 10CV Expell Linear 0-1 mM Tris/ 7 ~6 pH 7.5, 2MNaCl SP1 Gradient over 40 CV Acetate 6.0 mS/cm Protamine Linear 0-1 mMTris/ 7.5 ~6 pH 7.5, 2M NaCl, Sulfate Gradient over 40 CV Acetate 5.9 6MmS/cm Guanidine HCl mAb X Expell Two- (1): 0.5 mM, Tris/ 6 6.1 pH 6, 6.32M NaCl SP1 step 22CV; Acetate mS/cm (2): 2 mM, 12CV Protamine Two- (1):Tris/ 6 ~6 pH 6, 6.3 2M NaCl Sulfate step 0.35 mM, Acetate mS/cm 10CV;(2): 0.5 mM, 10CV

TABLE 3 Processing conditions for Capto MMC one-step displacementchromatography Equilibration/Wash/Displacing Displacer bufferConcentration Buffer Conductivity Loading Molecule Displacer (mM) SystempH (mS/cm) Conditions Regeneration CIP Adalimumab Protamine 0.25-0.5Tris/ 7-7.5 ~6 pH 7.5, 5.3- 2M NaCl, 0.5N NaOH + Sulfate Acetate 6.1mS/cm 6M 0.5M KCl Guanidine HCl mAb X Protamine 0.25-0.5 Tris/ 7-7.7 ~6pH 7-7.7, 2M NaCl, Sulfate Acetate 5.9-6.5 6M mS/cm Guanidine HCl mAb YProtamine 0.25-0.5 Tris/ 5-5.5 ~6.5 pH 5.5, 5.2- 2M NaCl, SulfateAcetate 5.6 mS/cm 6M Guanidine HCl

Example 1 Displacement Chromatography Performances of Expell SP1™ forAdalimumab on Poros XS Resin

Expell SP1™ is a low molecular weight quaternary ammonium salt thatexhibited pronounced displacement effect for Adalimumab on Poros XSresin under selected sets of operating conditions. The feed materialused for this set of experiments contained about 20-25% total AR, ofwhich 2-5% was AR1 and 18-20% AR2. The results for this system are shownin the following sections.

A representative, desired displacement chromatographic profile is shownin FIG. 1 a (solid line). In this experiment, the column wasequilibrated with a pH 7 Tris/acetate buffer (6.4 mS/cm), loaded with apre-adjusted protein A eluate feed (pH 7.5, 6.3 mS/cm, ˜3.4 g/L) to ˜40g/L resin loading level, followed by EQ buffer wash and thendisplacement process using 1 mM Expell SP1™ in the pH 7 EQ buffer. Theextended, square shape UV280 “elution” profile indicated establishing aproper displacement train and thus a degree of separation of the feedcomponents can be realized.

FIG. 2 illustrates the CEX-HPLC chromatograms for several samples takenalong this well-established displacement UV trace. Clearly, the variantspecies were rearranged during the displacement process according totheir respective binding affinity to the resin: AR1 was enriched in theforemost of the displacement train followed by AR2, Lys 0, Lys 1 and Lys2 in order.

FIG. 3 shows the distribution of each variant species in all thecollected sample fractions. The acidic species were enriched in theearlier fractions compared to the Lys variants. By excluding thoseearlier fractions the product pool AR level will be reduced relative tothat in the feed. This is reflected in FIG. 4 which plots the reductionof total AR (i.e., AR1+AR2) and AR1 level versus cumulative productyield. At a yield of ˜75%, the total AR % was reduced by 11.7% and AR1%by 4.2% under this set of condition. Along with the removal of ARspecies, the product pool lysine variant species distribution profilewas also modulated. As shown in Table 4, below, the ratio of Lys 0species to the lysine variant sum decreased from 0.67 to 0.62; the ratioof Lys 1 species to the lysine variant sum increased from 0.24 to 0.28;and the ratio of Lys 2 species to the lysine variant sum increased from0.08 to 0.1. The lysine prolife can be further altered by poolingdifferent fractions from the displacement chromatography process.

Varying the processing conditions such as the buffer pH and displacerconcentration can modulate the shape of the displacement chromatogramand hence the separation performance. In an extreme case, thechromatogram more or less resembles the typical elution “peak” profilewithout incurring the separation of variant species (FIGS. 1A and 1B).Interestingly, this occurs at stronger binding conditions; for instance,the conditions corresponding to FIG. 1B is pH 6.1 and ˜2 mS/cm forequilibration, loading, wash, and displacement. Without being bond bytheory, the lack of variant separation under such conditions may be dueto the diminishing difference in binding affinity of each species andthus the selectivity by the displacer.

The effect of Expell SP1™ concentration on Adalimumab AR reduction wasmeasured in pH 7.5 Tris/Acetate buffer, as shown in FIG. 5. The sameequilibration/wash and feed loading conditions were used for all theruns here. Increasing Expell SP1™ concentration from 0.5 to 3 mMdecreased ΔAR % from 8.9% to 3.5% at similar product yield ˜75%.Controlling the Expell SP1™ concentration within 2 mM will consistentlyachieve ≧6% AR % reduction.

The effect of displacing buffer pH on AR reduction for Adalimumab wasmeasured at 1 mM Expell SP1™ concentration in the Tris/Acetate buffer,as shown in FIG. 6. In this set of experiments, the column wasconditioned with an EQ buffer at the respective displacing buffer pH,and then loaded with protein feed at pH 7.5 and ˜6 mS/cm followed by abrief EQ buffer wash before starting the displacement step. The bufferpH significantly impacts AR clearance in pH range of 6-8. At similaryield (˜75%), the maximal reduction in AR level (˜12%) is seen at pH 7.Despite such pH-dependency, the majority of the conditions here (pH 6.5to 7.8) gave at least 5% AR removal in final product pool.

In the aforementioned experiments, one displacing buffer was used toachieve the protein variant separation. It was observed that, relativelylower displacer concentration gives better separation but tends toelongate the process due to substantial increase in the requireddisplacing buffer volume. For instance, when using 0.5 mM Expell SP1™ ina pH 7 displacing buffer (Table 1), the displacement phase requires 44column volumes (CV) of this buffer for completion. To accelerate theoperation without affecting the acidic species separation, a two-stepdisplacement process was explored at this pH condition. In the exampleprovided here, the displacement process was started with 0.5 mM ExpellSP1™ at pH 7 and continued for 25 CV, followed by 20 CV of 2 mM ExpellSP1™ solution at the same pH. Under such conditions, the proteindisplacement profile was completed in a total of 33 CV which is 25% lessthan that required for one-step displacement process, thus significantlyshortening the process.

FIG. 7 shows the reduction of AR % versus product yield for theaforementioned two-step displacement run. The net total AR level inproduct pool was reduced by 6.6% at ˜75% yield. In contrast to theconventional use of a single displacing solution consisting of a singledisplacer at a defined concentration, herein the AR clearance wasachieved by excluding the AR-enriched early fractions as induced by 0.5mM Expell SP1™ displacement, while the higher Expell SP1™ concentrationwas used to accelerate the displacement of the remainder proteins offthe solid phase. In light of this unexpected similar product quality andyield results, step-gradient displacement schemes are considered to beadvantageous over conventional strategies.

Besides the two-step displacement scheme, a linear gradient displacementmethod was also tested for the Adalimumab charge variant separation. Asdetailed in Table 2, after the feed loading at pH 7.5 (˜6 mS/cm), thecolumn was briefly washed with the equilibration buffer (pH 7, ˜6 mS/cm)and then started with a 40 CV linear gradient from the EQ buffer to a 1mM Expell SP1™ displacing buffer (which was made from the EQ buffer).Under such condition, the displacement profile matured within this 40 CVgradient. The product eluate was pooled by excluding the first a fewfractions. In this case, the net AR % decreased by 6.8% at a productrecovery of 72%.

Apart from acidic species, other product- or process-related impuritiescan be effectively separated by Poros XS displacement chromatographyusing Expell SP1™ as the displacer. FIG. 8 shows the separation ofaggregates, monomer and fragments in Adalimumab sample fractionsobtained from a one-step displacement experiment using 1 mM Expell SP1™,pH 7 buffer. It should be noted that the last two fractions from thisrun were not collected, therefore the increased aggregate levels at theend of the displacement train was not fully exemplified here.Interestingly, the early fractions which contained elevated acidicspecies also showed enriched aggregates, indicating that this populationof aggregates may consist of more acidic species, or the acidic specieshas higher propensity to form aggregates. As summarized in Table 4, theaggregate level in the product pool (at ˜75% yield) was reduced from thefeed level 1.16% to 0.11% and the fragment level down to 0.04% alongwith significant reduction in the AR concentration. In addition to thestandard method, the linear gradient displacement run also showedaggregate reduction from the feed level of 0.9% to about 0.2% in finalproduct.

FIG. 9 shows the distribution of HCP in the Adalimumab displacementtrain coming off the Poros XS column. Relatively higher level of HCP wasobserved at both ends of the train, due to their diverse chargecharacteristics and associated binding strength. The final product poolHCP level was reduced to 5 ng/mg from the starting feed, representingapproximately 50-fold reduction.

TABLE 4 Step yield & product quality in Adalimumab before and afterPoros XS displacement chromatography using Expell SP1 ™ (pH 7, 1 mMExpell SP1 ™) Yield AR1 AR2 Lys Lys 0 Lys 1 Lys 2 HMW Monomer LMW HCP %% % Sum % % % % % % % (ng/mg) Feed — 4.3 17.8 77.9 52.5 19.0 6.5 1.1698.57 0.27 267 Product 74 0.1 10.3 89.6 55.5 24.8 9.3 0.11 99.85 0.04 5pool

Example 2 Displacement Chromatography Performance of Protamine Sulfatefor Adalimumab on Poros XS Resin

Protamine sulfate, a cationic peptide with molecule weight ˜5.1 kD, wasalso evaluated as a cation exchange displacer for Adalimumab on Poros XSresin under various operating conditions. The feed material used forthis set of experiments contained about 17-24% total AR, of which 3-6%was AR1 and 14-19% AR2. The results for this system are illustrated inthe following sections.

FIG. 10 shows the distribution of charge variant species in samplefractions collected from a well established displacement process inducedby protamine sulfate. In this experiment, the column was equilibratedwith a pH 7.5 Tris/acetate buffer (5.6 mS/cm), loaded with apre-adjusted protein A eluate feed (pH 7.5, 5.4 mS/cm, 5.2 g/L) to 39g/L resin loading level, followed by a brief EQ buffer wash and thendisplacement process using 0.5 mM protamine sulfate dissolved in the pH7.5 EQ buffer. Similar to the Expell SP1™ displacement profile (FIG. 3),the charge variants were enriched at different locations of thedisplacement train and were peaked in the order of AR1, AR2, Lys0, Lys1and Lys2. The cumulative AR % reduction as a function of product yieldis illustrated in FIG. 11. A 6-8% decrease in the total AR level can beobtained at a yield of 75-85% under this set of condition. The actuallevels of AR1, AR2 and total lysine variant species (i.e., Lys 0+Lys1+Lys 2) for the feed and the final product pool are shown in Table 5.

The effect of protamine sulfate concentration on Adalimumab AR reductionwas measured in pH 7.5 Tris/Acetate buffer, as shown in FIG. 12. Thesame equilibration/wash and feed loading conditions as described abovewere used for all the runs here. At similar yield (˜75%), the total AR %was reduced by approximately 7-8% when using 0.25 to 2 mM protaminesulfate. This broad concentration range reflects the robustness ofcharge variant separation by protamine sulfate displacement process.

The effect of displacing buffer pH on AR clearance for Adalimumab wasmeasured at 0.5 mM protamine sulfate concentration in Tris/Acetatebuffer. In this set of experiments, the column was conditioned with anEQ buffer at the respective displacing buffer pH, loaded with proteinfeed at pH 7.5 and ˜6 mS/cm followed by a brief EQ buffer wash beforestarting the displacement phase. As shown in FIG. 13, the extent of ARreduction increases significantly as pH varies from 6.5 to 7.5. Over 6%decrease in AR level can be achieved at pH 7.5 with a product yield˜75%.

The two-step displacement scheme was also tested with protamine sulfate.In one experiment, the displacement process consists of 10 CV of 0.25 mMprotamine and 10 CV of 2 mM protamine at pH 7.5 (Table 2). The proteindisplacement profile was completed in a total of 13 CV, which is about11 CV or almost 2 fold shorter than that in the one-step displacementprocess with 0.25 mM protamine sulfate. The reduction of AR % versusproduct yield is shown in FIG. 14. The total AR level in product poolwere reduced by ˜8% at ˜75% yield, which is comparable to that achievedby the one-step displacement process using 0.25 mM protamine sulfate.

The linear gradient displacement scheme was also evaluated withprotamine sulfate on Poros XS resin for Adalimumab charge variantseparation. As summarized in Table 2, after the feed loading at pH 7.5(5.9 mS/cm), the column was briefly washed with the equilibration buffer(pH 7.5, ˜6 mS/cm) and then started with a 40 CV linear gradient fromthe EQ buffer to a 1 mM protamine sulfate displacing buffer (which wasmade from the EQ buffer). FIG. 15 shows the cumulative ΔAR % versusyield from this run. At a product yield of 75.6%, the total AR % wasreduced from the feed level of 21.3% to 12.1%.

Protamine sulfate displacement chromatography also demonstratedsignificant clearance of aggregates, fragments and HCP. FIG. 16exemplifies the size variant profiles of Adalimumab from the sameexperiment described above (i.e., 0.5 mM protamine sulfate, pH 7.5,one-step displacement run). As expected, the fragments were mostlyenriched at the front while the aggregates primarily resided at the backof the train. Similar to that shown in FIG. 8, a subpopulation of theaggregates was also observed in the displacement front; in addition, aportion of fragments was noticed at the tail. Table 5 compares thelevels of aggregates, fragments and HCP in final product pool (at ˜75%yield) relative to the feed.

TABLE 5 Step yield & product quality in Adalimumab before and afterPoros XS displacement chromatography using protamine sulfate Yield AR1AR2 Lys HMW Monomer LMW HCP % % % Sum % % % % (ng/mg) Feed — 4.1 16.979.0 0.8 98.0 1.2 153 Product 73 1.3 13.1 84.7 0.3 99.6 0.1 14 pool

Example 3 Displacement Chromatography Performance of Expell SP1™ for mAbX on Poros XS Resin

The displacement separation performance of Expell SP1™ was assessed formAb X on the Poros XS resin. A purified mAb X drug substance was used inthis study, which contained about 16-17% acidic species and 12-14% basicspecies.

A representative set of mAb X charge variant separation profiles areshown in FIGS. 17 and 18. In this experiment, the Poros XS column wasloaded with 40 g/L of mAb X at pH 6, 6 mS/cm Tris/Acetate bindingcondition, and was displaced using 1 mM Expell SP1™ in a pH 6, ˜6 mS/cmbuffer. The specific conditions are detailed in Table 1. Pronouncedenrichment and separation of acidic, main and basic species wereachieved, with AR % reduced by 9.4% at 76% yield.

The effect of Expell SP1™ concentration on AR reduction for mAb X wasmeasured in the pH 6 Tris/Acetate buffer. As shown in FIG. 19,increasing the Expell SP1™ concentration from 0.5 to 2 mM decreased theΔAR % for mAb X from 9.8% at 81% yield to 7.9% at 69% yield.

The two-step displacement scheme was evaluated for mAb X. As shown inTable 2, the displacement process comprised of 22 CV of 0.5 mM ExpellSP1™ and 12 CV of 2 mM Expell SP1™ at pH 6. The protein displacementprofile was completed within 30 CV of total displacing buffer volume,which was 30% less than that required for one-step displacementseparation. The reduction of AR % versus product yield is shown in FIG.20. The total AR % in product pool was reduced by ˜9% at ˜75% yield,again comparable to that obtained with one-step displacement processusing 0.5 mM Expell SP1™ buffer.

Example 4 Displacement Chromatography Performance of Protamine Sulfatefor mAb X on Poros XS Resin

Protamine sulfate was also evaluated for separating acidic species formAb X on Poros XS resin. The feed material for this set of experimentscontained about 12-16% acidic and 12-13% basic species. The results forthis system are shown in the following sections.

A representative set of variant separation profiles are shown in FIGS.21 and 22. In this experiment, the Poros XS column was loaded with ˜36g/L of mAb X at pH 6, 6.5 mS/cm binding condition, and was displacedusing 0.25 mM protamine sulfate in a pH 6, 6.5 mS/cm Tris/Acetatebuffer. The specific conditions are detailed in Table 1. Pronouncedenrichment and separation of acidic, main and basic species wereachieved, with AR level reduced by 6% at 75% yield.

The effect of protamine sulfate concentration on mAb X AR reduction wasmeasured in a pH 6 Tris/Acetate buffer, as shown in FIG. 23. In thiscase, the protamine sulfate concentration strongly affects the ARclearance in a relatively small protamine concentration range (i.e.,from 0.35 to 0.5 mM). Nevertheless, over 8% of AR reduction can beachieved at pH 6 for mAb X at acceptable yield (≧70%).

The two-step displacement scheme was evaluated for mAb X with protaminesulfate. In this experiment, the displacement process comprised of 10 CVof 0.35 mM Expell and 10 CV of 0.5 mM Expell at pH 6 (see Table 2). Theprotein displacement profile was completed in ˜15 CV of total displacingbuffer volume, representing ˜26% reduction of buffer volume relative tothe one-step displacement operation. The reduction of AR % versusproduct yield is shown in FIG. 24. The product pool AR level was reducedby ˜6% at ˜75% yield.

The mAb X BDS has about 0.74% of aggregates, which can be furtherreduced during the protamine sulfate displacement process. FIG. 25 showsthe size variant profiles of mAb X as displaced by a 0.25 mM protaminesulfate, pH 6 buffer (i.e., the one-step displacement run shown in FIGS.21 and 22). The aggregates were all enriched at the end of thedisplacement train, which differs from the observations with Adalimumab.Using the same product pooling strategy based on AR reduction, themonomer level was enhanced to 99.9% (Table 6).

TABLE 6 Step yield & product quality in mAb X before and after Poros XSdisplacement chromatography using protamine sulfate Yield % Acidic %Main % HMW % Monomer % Feed — 12.3 75.7 0.7 99.3 Product pool 79 4.780.8 0.1 99.9

Example 5 Displacement Chromatography Performance of Expell SP1™ for mAbY on Poros XS Resin

The displacement separation performance of Expell SP1™ was furtherassessed for mAb Y on Poros XS resin. The mAb Y has a pI of 7-7.5, muchlower than Adalimumab and mAb X. An mAb Y protein A eluate was used inthis study, which contained about 22% acidic species and 15% basicspecies.

An appropriate set of displacement conditions for mAb Y is shown inTable 1. The equilibration, wash and displacement buffers are all at pH5 with conductivity around 6 mS/cm. The 0.5 mM Expell SP1™ buffergenerated the desired displacement profile. The sample fractions fromthis run were analyzed by cation exchange HPLC. FIGS. 26 and 27 indicatethe distribution of charge variant species and the cumulative ΔAR %versus product yield, respectively. A 6.6% decrease in AR % was observedat 74% yield under such condition.

Example 6 Displacement Chromatography Performance of Protamine Sulfatefor Adalimumab on Capto MMC (Multimodal) Resin

Capto MMC™ is a mixed mode resin based on weak cation-exchange andhydrophobic interaction mechanism. Its capability for acidic species andaggregates removal by displacement chromatography was assessed here. TheAdalimumab feed material for this set of experiments contained about20-21% total AR. The results for protamine sulfate system are shown inthe following sections.

A representative set of variant separation profiles are shown in FIGS.28 and 29. In this experiment, the Capto MMC column was equilibratedwith a 140 mM Tris/acetate, pH 7 buffer (˜5.7 mS/cm), loaded with ˜34g/L of Adalimumab at pH 7.5 and 5.3 mS/cm binding condition, brieflywashed with EQ buffer and then displaced with 0.35 mM protamine sulfatein the pH 7 EQ buffer. A typical displacement chromatogram was generatedunder such experimental condition. As shown in FIG. 10, Capto MMC alsoshowed enrichment of each variant in the train, yielding total ARreduction of ˜4% at ˜75% yield. The shape of the ΔAR % versus yieldcurve (FIG. 29) differs from that given by the Poros XS resin, possiblydue to stronger binding of each protein species (related to secondarymode of interaction) by this mixed mode ligand.

The buffer pH and protamine sulfate concentrations were varied to assessthe overall AR clearance by Capto MMC displacement chromatography. Table7 summarized the results for three runs. Overall, 3-5% of AR reductioncan be achieved for Adalimumab when using protamine sulfate as adisplacer for Capto MMC resin.

TABLE 7 AR removal for Adalimumab by Capto MMC displacementchromatography using protamine sulfate Protamine Run EQ/Displacing Conc.No. buffer pH Load pH (mM) Yield (%) ΔAR % 1 7 7 0.5 75 3.1 2 7 7.5 0.3575 4.0 3 7.25 7.5 0.25 78 5.2

The clearance of aggregates by Capto MMC displacement chromatography isillustrated in Table 8. The same operating conditions as described forobtaining the results in Table 7 were used here. The product poolmonomer level was enhanced from 98.8% in the feed to 99.4% withaggregates reduced from 1.0% to 0.5%.

TABLE 8 Step yield & product quality in Adalimumab before and afterCapto MMC displacement chromatography using protamine sulfate Yield AR1AR2 Lys HMW Monomer LMW % % % Sum % % % % Feed — 4.4 16.5 79.1 1.0 98.80.2 Product 75 2.5 14.4 83.1 0.5 99.4 0.1 pool

Example 7 Displacement Chromatography Separation of Protamine Sulfatefor mAb X on Capto MMC Resin

Protamine sulfate was evaluated for removing mAb X acidic species onCapto MMC resin. The same feed material as shown in Example 4 was usedfor this set of experiments. As detailed in Table 3, the pH andprotamine concentrations were varied in order to generate desireddisplacement profile. One representative set of working condition is toload Capto MMC column with 40 g/L of mAb X at pH 7.5 and ˜6 mS/cm, andto use 0.25 mM protamine sulfate in the pH 7.5, ˜6 mS/cm EQ buffer fordisplacement (Table 3). The separation of charge variants and ARreduction as a function of product recovery are shown in FIGS. 30 and31, respectively. In this case, 3-5% of AR reduction was resulted atproduct yield of 70-90%.

The effect of protamine sulfate concentration on mAb X AR reduction byCapto MMC resin was measured in a pH 7, 6 mS/cm Tris/Acetate buffer, asshown in Table 9. Varying the protamine concentration from 0.25 mM to0.5 mM had very little effect on ΔAR % and product yield, which is quitedifferent from the observations with the Poros XS resin (FIG. 23). Themixed mode MMC resin gave over 3% AR clearance under such selectedconditions.

TABLE 9 Reduction of AR level by Capto MMC displacement chromatographyfor mAb X at different protamine sulfate concentrations Expellconcentration (mM) Yield (%) ΔAR (%) 0.25 75 3.4 0.5 77 3.3

The effect of displacing buffer pH on AR clearance for mAb X wasmeasured at 0.25 mM protamine sulfate concentration in Tris/Acetatebuffer. In this set of experiments, the column was conditioned with anEQ buffer at the respective displacing buffer pH, loaded with proteinfeed at the same pH and ˜6 mS/cm followed by a brief EQ buffer washbefore starting the displacement phase. As shown in FIG. 32, ΔAR %increases from 3.3 to 6.5% as pH varies from 7 to 7.7.

The Capto MMC resin also removes aggregates during protamine sulfatedisplacement chromatography. Table 10 shows the level of charge and sizevariants of mAb X in product eluate when using 0.5 mM protamine sulfate,pH 7.5 (˜6 mS/cm) displacing buffer for separation. The product poolmonomer level was enhanced from 98.7% in the feed to 99.2% withaggregate and fragment levels reduced by 50% and 28%, respectively.

TABLE 10 Step yield & product quality for mAb X before and after CaptoMMC displacement chromatography using protamine sulfate Yield AcidicMain HMW Monomer LMW % % % % % % Feed — 16.8 74.0 0.6 98.7 0.7 Product77 12.4 71.8 0.3 99.2 0.5 pool

Example 8 Displacement Chromatography Separation of Protamine Sulfatefor mAb Y on Capto MMC Resin

Protamine sulfate was also evaluated for removing mAb Y acidic specieson Capto MMC resin. The same feed material as shown in Example 5 wasused for the experiments here. Two sets of conditions were evaluatedhere. In one experiment, the equilibration, wash and displacementbuffers were adjusted to pH 5.5 and ˜6.5 mS/cm, and the displacementbuffer contained 0.25 mM protamine sulfate. In the other experiment, allthose buffers were adjusted to pH 5, ˜6.5 mS/cm and the protaminesulfate concentration was 0.5 mM. In both runs the feed was adjusted topH 5.5, 5.2-5.5 mS/cm and loaded to the Capto MMC column at ˜40 g/Lloading level. As shown in Table 11, both sets of conditions resulted inAR % decrease by 5-7% in product pool. In addition, significantaggregates and fragments reduction was achieved with the same productpooling strategy.

TABLE 11 Step yield & product quality for mAb Y before and after CaptoMMC displacement chromatography using protamine sulfate Mono- YieldAcidic Main HMW mer LMW Conditions Sample % % % % % % pH 5.5, Feed —16.4 75.8 8.5 91.0 0.5 0.25 mM Product 76 9.8 79.4 2.0 97.6 0.4Protamine pool pH 5, Feed — 20.9 72.8 6.3 93.2 0.5 0.5 mM Product 7515.9 81.1 1.6 98.2 0.2 Protamine pool

Summary of Examples 1-8

Examples 1-8 above, demonstrate the use of cation exchange and mixedmode displacement chromatography for effectively reducing acidic speciesalong with various other impurities from different monoclonal antibodyfeed streams. Under appropriate (or relatively weak) binding conditions,cationic molecules with high affinity for a CEX or multimodal ligand(such as Expell SP1™ and protamine sulfate) can induce the formation ofcharge variant displacement train, wherein the acidic population isenriched in the front followed by the main isoform, and, thereafter, thebasic population. Thus, in certain embodiments, exclusion of thoseearlier fractions from the remainder eluate results in an AR-reducedproduct. Alternatively, exclusion of the fractions following the mainisoform results in a Lys variant- or basic species-reduced product.

Also demonstrated in the preceding experiments is the fact that theoperating pH and displacer concentration can strongly affect thedisplacement profile and as a result the charge variant, productaggregate, product fragment, and HCP clearance profile. The selection ofa particular operating regime with regard to charge variant reductiondepends, in general, on the specific protein-resin-displacer system. Forexample, for Adalimumab, significant AR reduction can be achieved usinga displacing buffer with pH in the range of 6-8 with displacerconcentration as low as 0.25-0.5 mM. The total AR level (%) inAdalimumab product pool can be reduced by over 10% with an acceptableprocessing yield (≧75%) from a CEX displacement chromatography process,or 4-7% from a mixed mode displacement chromatography process. Alongwith acidic species, other product variants or process impurities suchas basic species, aggregates, fragments and HCP can be selectivelycollected or reduced to meet the quality requirements. In addition tothe surprisingly effective preparative scale standard one-stepdisplacement operation, unconventional displacement separation schemesare shown above to have unexpected properties, including two-stepdisplacement chromatography and linear gradient displacementchromatography, which can significantly reduce buffer volumes andshorten the processing time without compromising the charge variant,product aggregate, product fragment, and HCP clearance at a given yieldtarget.

The instant invention provides a method for reducing acidic species fora given protein of interest. For example, adalimumab was prepared usinga combination of AEX and CEX technologies to produce a Low-AR andHigh-AR sample with a final AR of 2.5% and 6.9%, respectively. Bothsamples were incubated in a controlled environment at 25° C. and 65%relative humidity for 10 weeks, and the AR measured every two weeks.FIG. 23 shows the growth of AR for each sample over the 10 weekincubation. It is evident from FIG. 23 the growth rate of AR is linearand similar between both the Low-AR and High-AR samples. Based on theseresults, the reduced AR compositions of the invention can be stored3-fold longer before reaching the same AR level as the High—AR sample.These surprising results are very beneficial for storage, handling, anduse of an antibody or other protein for therapeutic use.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1. A method for producing a low acidic species adalimumab composition,the method comprising: (a) contacting a first composition comprisingadalimumab with a chromatography media, wherein the first compositioncomprises more than 10% total acidic species of adalimumab, wherein theacidic species of adalimumab have a net negative charge relative toadalimumab main species, wherein the acidic species of adalimumabcomprise species selected from the group consisting of charge variants,structure variants, fragmentation variants, and any combinationsthereof, wherein the acidic species of adalimumab do not includeprocess-related impurities selected from the group consisting of hostcell proteins, host cell nucleic acids, chromatographic materials andmedia components, and wherein the adalimumab binds to the chromatographymedia; (b) displacing the adalimumab bound to the chromatography mediawith a displacing buffer; and (c) collecting a second compositioncomprising the displaced adalimumab, wherein the second compositioncomprises less than 10% total acidic species of adalimumab, therebyproducing a low acidic species adalimumab composition.
 2. The method ofclaim 1, wherein the pH of the displacing buffer is lower than theisoelectric point of adalimumab.
 3. The method of claim 1, wherein thedisplacing buffer carries a positive charge.
 4. The method of claim 1,wherein the conductivity of the displacing buffer is about 2 mS/cm toabout 20 mS/cm.
 5. The method of claim 1, wherein one displacing bufferis used.
 6. The method of claim 1, wherein displacing the adalimumabbound to the chromatography media comprises using a first displacingbuffer followed by using a second displacing buffer.
 7. The method ofclaim 1, wherein displacing is achieved using linear displacement. 8-30.(canceled)
 31. The method of claim 1, wherein the second compositioncomprises less than 9% total acidic species of adalimumab.
 32. Themethod of claim 1, wherein the second composition comprises less than 8%total acidic species of adalimumab.
 33. The method of claim 1, whereinthe second composition comprises less than 7% total acidic species ofadalimumab.
 34. The method of claim 1, wherein the second compositioncomprises less than 6% total acidic species of adalimumab.
 35. Themethod of claim 1, wherein the second composition comprises less than 5%total acidic species of adalimumab.
 36. The method of claim 1, whereinthe second composition comprises less than 4.5% total acidic species ofadalimumab.
 37. The method of claim 1, wherein the second compositioncomprises less than 4% total acidic species of adalimumab.
 38. Themethod of claim 1, wherein the second composition comprises less than 3%total acidic species of adalimumab.
 39. The method of claim 1, whereinthe second composition comprises less than 1.4% total acidic species ofadalimumab.
 40. The method of claim 1, wherein the displacing buffercomprises protamine sulfate.
 41. The method of claim 1, wherein thedisplacing buffer comprises a quaternary ammonium salt.
 42. (canceled)43. (canceled)
 44. The method of claim 1, wherein the chromatographymedia is an anion exchange adsorbent material.
 45. The method of claim1, wherein the chromatography media is a cation exchange adsorbentmaterial.
 46. The method of claim 45, wherein the cation exchangeadsorbent material is a CEX membrane adsorber.
 47. The method of claim45, wherein the cation exchange adsorbent material is a CEX resin. 48.The method of claim 1, wherein the chromatography media is a mixed modemedia comprising cation exchange and hydrophobic interaction functionalgroups.
 49. (canceled)
 50. The method of claim 1, wherein thechromatography media is a mixed mode media selected from the groupconsisting of a cation exchange-based mixed mode resin and acation-exchange-based mixed mode membrane adsorber.
 51. (canceled) 52.(canceled)
 53. The method of claim 1, wherein the displacing buffercomprises protamine sulfate, a quaternary ammonium salt, apolyelectrolyte, a polysaccharide, a low-molecular-mass dendrimer, anamino acid, a peptide, an antibiotic, a polyaromatic polyanioniccompound, or an aminoglycosidepolyamine.
 54. The method of claim 53,wherein the concentration of the protamine sulfate, quaternary ammoniumsalt, polyelectrolyte, polysaccharide, low-molecular-mass dendrimer,amino acid, peptide, antibiotic, polyaromatic polyanionic compound, oraminoglycosidepolyamine in the displacing buffer is 0.1 mM to 10 mM. 55.The method of claim 1, wherein displacing is achieved using a two-stepdisplacement.
 56. The method of claim 1, wherein displacing is achievedusing a multiple-step displacement.